Polynucleotides for use as tags and tag complements in the detection of nucleic acid sequences

ABSTRACT

A family of minimally cross-hybridizing nucleotide sequences and their use in the detection of nucleic acid sequences is described. Specifically described is the use of two distinct families of 210 and 1168 24mers in the detection of nucleic acid sequences.

FIELD OF THE INVENTION

This invention relates to the use of families of oligonucleotides for use as tags, for example, in the sorting of molecules, identification of target nucleic acid molecules or for analyzing the presence of a mutation or polymorphism at a locus of each target nucleic acid molecule.

BACKGROUND OF THE INVENTION

Single-nucleotide polymorphisms (SNPs) are the most common form of genetic polymorphism. This, coupled with their potential as functional variants, has produced a great deal of interest in SNPs both as pharmacogenetic indicators and as markers for mapping genes for complex diseases. A large number of SNPs have already been identified with >21,000 entries on the NCBI's SNP database alone. Many recent studies have focused on identifying polymorphisms that lie in the coding sequence of potential candidate genes for common diseases. The ability to genotype this abundant source of variation rapidly and accurately is becoming an ever more important goal in the genetics community. A variety of technologies have the potential to transfer to high-throughput genotyping laboratories. These include 5′ exonuclease assays, such as TaqMan (Livak et al. 1995), molecular beacons (Tyagi et al. 1996), dye-labeled oligonucleotide ligation (DOL) (Chen et al. 1998), oligonucleotide-ligation assays (OLAs) (To be et al. 1996), minisequencing (Chen and Kwok 1997; Pastinen et al. 1997), microarray technology (Hacia et al. 1998; Wang et al. 1998), mass spectroscopy (Ross et al. 1998) and the scorpions assay (Whitcombe et al. 1999). However, no single chemistry has gained acceptance as the technology of choice. A suitable method for such applications must be accurate and homogenous, develop a robust, easily interpretable signal, and be flexible enough to extend to novel foci with little optimization. These features will lend the technology to automation.

Third Wave Technologies, Inc., has developed a new mutation detection method referred to as the Invader Assay. The Invader Assay is based on a novel linear signal amplification technology that requires specific hybridization of two “overlapping” oligonucleotides and subsequent recognition and cleavage of this structure by the Cleavase enzyme. Cleavases are bacterial enzymes that cleave unpaired DNA strands or “flaps” near a nick, for instance when the 5′ end of a sequence is displaced by the 3′ end of an elongating upstream oligonucleotide. Enzymes with this so-called flap endonuclease activity typically excise the redundant 5′ “flap” of the downstream oligonucleotide, leaving a simple nick to be repaired by lipases. The excised “flap” is subsequently detected by one of several methods commonly known in the art. Cleavases have stringent requirements relative to the structure formed by such overlapping DNA sequences, and can be used to specifically detect single base pair mismatches immediately upstream of the cleavage site on the downstream DNA strand. Thermostable cleavages permit reactions to be performed at temperatures sufficiently high to promote turnover and consequent signal amplification without the need for temperature cycling.

While the Invader Assay offers exquisite specificity, its use in the detection of multiple distinct target nucleic acids in a single experiment i.e., multiplexing, is limited. This is because if the Invader Assay is to be used in a high-throughput gene microarray format, the most efficient method of detecting the excised “flap” sequence is to capture the sequence by hybridization to its complementary nucleic acid sequence attached to a solid phase support.

Working in a highly parallel hybridization environment requiring specific hybridization imposes very rigorous selection criteria for the design of families of oligonucleotides that are to be used. The success of these approaches is dependent on the specific hybridization of a probe and its complement. Problems arise as the family of nucleic acid molecules cross-hybridise or hybridise incorrectly to the target sequences. While it is common to obtain incorrect hybridization resulting in false positives or an inability to form hybrids resulting in false negatives, the frequency of such results must be minimized. In order to achieve this goal certain thermodynamic properties of forming nucleic acid hybrids must be considered. The temperature at which oligonucleotides form duplexes with their complementary sequences known as the T. (the temperature at which 50% of the nucleic acid duplex is dissociated) varies according to a number of sequence dependent properties including the hydrogen bonding energies of the canonical pairs A-T and G-C (reflected in GC or base composition), stacking free energy and, to a lesser extent, nearest neighbour interactions. These energies vary widely among oligonucleotides that are typically used in hybridization assays. For example, hybridization of two probe sequences composed of 24 nucleotides, one with a 40% GC content and the other with a 60% GC content, with its complementary target under standard conditions theoretically may have a 10° C. difference in melting temperature (Mueller et al., Current Protocols in Mol. Biol.; 15, 5:1993). Problems in hybridization occur when the hybrids are allowed to form under hybridization conditions that include a single hybridization temperature that is not optimal for correct hybridization of all oligonucleotide sequences of a set. Mismatch hybridization of non-complementary probes can occur forming duplexes with measurable mismatch stability (Santalucia et al., Biochemistry; 38: 3468-77, 1999). Mismatching of duplexes in a particular set of oligonucleotides can occur under hybridization conditions where the mismatch results in a decrease in duplex stability that results in a higher T_(m) than the least stable correct duplex of that particular set. For example, if hybridization is carried out under conditions that favor the AT-rich perfect match duplex sequence, the possibility exists for hybridizing a GC-rich duplex sequence that contains a mismatched base having a melting temperature that is still above the correctly formed AT-rich duplex. Therefore, design of families of oligonucleotide sequences that can be used in multiplexed hybridization reactions must include consideration for the thermodynamic properties of oligonucleotides and duplex formation that will reduce or eliminate cross hybridization behavior within the designed oligonucleotide set.

The development of such families of tags has been attempted over the years with varying degrees of success. There are a number of different approaches for selecting sequences for use in multiplexed hybridization assays. The selection of sequences that can be used as zipcodes or tags in an addressable array has been described in the patent literature in an approach taken by Brenner and co-workers. U.S. Pat. No. 5,654,413 describes a population of oligonucleotide tags (and corresponding tag complements) in which each oligonucleotide tag includes a plurality of subunits, each subunit consisting of an oligonucleotide having a length of from three to six nucleotides and each subunit being selected from a minimally cross hybridizing set, wherein a subunit of the set would have at least two mismatches with any other sequence of the set. Table II of the Brenner patent specification describes exemplary groups of 4mer subunits that are minimally cross hybridizing according to the aforementioned criteria. In the approach taken by Brenner, constructing non cross-hybridizing oligonucleotides, relies on the use of subunits that form a duplex having at least two mismatches with the complement of any other subunit of the same set. The ordering of subunits in the construction of oligonucleotide tags is not specifically defined.

Parameters used in the design of tags based on subunits are discussed in Barany et al. (WO 9731256). For example, in the design of polynucleotide sequences that are for example 24 nucleotides in length (24mer) derived from a set of four possible tetramers in which each 24mer “address” differs from its nearest 24mer neighbour by 3 tetramers. They discuss further that, if each tetramer differs from each other by at least two nucleotides, then each 24mer will differ from the next by at least six nucleotides. This is determined without consideration for insertions or deletions when forming the alignment between any two sequences of the set. In this way a unique “zip code” sequence is generated. The zip code is ligated to a label in a target dependent manner, resulting in a unique “zip code” which is then allowed to hybridise to its address on the chip. To minimise cross-hybridisation of a “zip code” to other “addresses”, the hybridization reaction is carried out at temperatures of 75-80° C. Due to the high temperature conditions for hybridization, 24mers that have partial homology hybridise to a lesser extent than sequences with perfect complementarity and represent ‘dead zones’. This approach of implementing stringent hybridization conditions for example, involving high temperature hybridization, is also practiced by Brenner et. al.

The current state of technology for designing non-cross hybridizing tags based on subunits does not provide sufficient guidance to construct a family of relatively large numbers of sequences with practical value in assays that require stringent non-cross hybridizing behavior.

Thus, while it is desirable to have, at once in a gene microarray format, a large number of “flap” molecules incorporated into the Invader Assay, the “flap” molecules should each be highly selective for its own complement sequence. While such an array provides the advantage that the family of molecules making up the grid is entirely of design, and does not rely on sequences as they occur in nature, the provision of a family of molecules, which is, sufficiently large and where each individual member is sufficiently selective for its complement over all the other zipcode molecules (i.e., where there is sufficiently low cross-hybridization, or cross-talk) continues to elude researchers.

SUMMARY OF THE INVENTION

The present invention relates to the use of one set of 210 and a second set of 1168 minimally cross-hybridizing oligonucleotide sequences for use in the Invader Assay. The incorporation of these sequences into one of the two probes, and subsequent structure dependent cleavage of the minimally cross-hybridizing sequences upon hybridization to the target nucleic acid molecule enables the Invader Assay to be used in the analysis of multiple gene n a gene microarray.

Using the method of Benight et al. a family of 100 sequences was obtained using a computer algorithm to have optimal hybridization properties for use in nucleic acid detection assays. The sequence set of 100 oligonucleotides was characterized in hybridization assays, demonstrating the ability of family members to correctly hybridize to their complementary sequences with an absence of cross hybridization. These are the sequences having SEQ ID NOs:1 to 100 of Table I. This set of sequences has been expanded to include an additional 110 sequences that can be grouped with the original 100 sequences as having non-cross hybridizing properties, based on the characteristics of the original set of 100 sequences. These additional sequences are identified as SEQ ID NOs:101 to 210 of the sequences in Table I. How these sequences were obtained is described below.

Variant families of sequences (seen as tags or tag complements) of a family of sequences taken from Table I are also part of the invention. For the purposes of discussion, families of tag complements will be described.

A family of complements is obtained from a set of oligonucleotides based on a family of oligonucleotides such as those of Table I. For illustrative purposes, providing a family of complements based on the oligonucleotides of Table I will be described.

Firstly, the groups of sequences based on the oligonucleotides of Table I can be represented as follows: TABLE IA Numeric sequences corresponding to word patterns of a set of oligonucleotides Sequence Identifier Numeric Pattern 1 1 4 6 6 1 3 2 2 4 5 5 2 3 3 1 8 1 2 3 4 4 1 7 1 9 8 4 5 1 1 9 2 6 9 6 1 2 4 3 9 6 7 9 8 9 8 10 9 8 9 1 2 3 8 10 9 8 8 7 4 3 1 10 1 1 1 1 1 2 11 2 1 3 3 2 2 12 3 1 2 2 3 2 13 4 1 4 4 4 2 14 1 2 3 3 1 1 15 1 3 2 2 1 4 16 3 3 3 3 3 4 17 4 3 1 1 4 4 18 3 4 1 1 3 3 19 3 6 6 6 3 5 20 6 6 1 1 6 5 21 7 6 7 7 7 5 22 8 7 5 5 8 8 23 2 1 7 7 1 1 24 2 3 2 3 1 3 25 2 6 5 6 1 6 26 4 8 1 1 3 8 27 5 3 1 1 6 3 28 5 6 8 8 6 6 29 8 3 6 5 7 3 30 1 2 3 1 4 6 31 1 5 7 5 4 3 32 2 1 6 7 3 6 33 2 6 1 3 3 1 34 2 7 6 8 3 1 35 3 4 3 1 2 5 36 3 5 6 1 2 7 37 3 6 1 7 2 7 38 4 6 3 5 1 7 39 5 4 6 3 8 6 40 6 8 2 3 7 1 41 7 1 7 8 6 3 42 7 3 4 1 6 8 43 4 7 7 1 2 4 44 3 6 5 2 6 3 45 1 4 1 4 6 1 46 3 3 1 4 8 1 47 8 3 3 5 3 8 48 1 3 6 6 3 7 49 7 3 8 6 4 7 50 3 1 3 7 8 6 51 10 9 5 5 10 10 52 7 10 10 10 7 9 53 9 9 7 7 10 9 54 9 3 10 3 10 3 55 9 6 3 4 10 6 56 10 4 10 3 9 4 57 3 9 3 10 4 9 58 9 10 5 9 4 8 59 3 9 4 9 10 7 60 3 5 9 4 10 8 61 4 10 5 4 9 3 62 5 3 3 9 8 10 63 6 8 6 9 7 10 64 4 6 10 9 6 4 65 4 9 8 10 8 3 66 7 7 9 10 5 3 67 8 8 9 3 9 10 68 8 10 2 9 5 9 69 9 6 2 2 7 10 70 9 7 5 3 10 6 71 10 3 6 8 9 2 72 10 9 3 2 7 3 73 8 9 10 3 6 2 74 3 2 5 10 8 9 75 8 2 3 10 2 9 76 6 3 9 8 2 10 77 3 7 3 9 9 10 78 9 10 1 1 9 4 79 10 1 9 1 4 1 80 7 1 10 9 8 1 81 9 1 10 1 10 6 82 9 6 9 1 3 10 83 3 10 8 8 9 1 84 3 8 1 9 10 3 85 9 10 1 3 6 9 86 1 9 1 10 3 1 87 1 4 9 6 8 10 88 3 3 9 6 1 10 89 5 3 1 6 9 10 90 6 1 8 10 9 6 91 5 9 9 4 10 3 92 2 10 9 1 9 5 93 10 10 7 2 1 9 94 10 9 9 1 8 2 95 1 8 6 8 9 10 96 1 9 1 3 8 10 97 9 6 9 10 1 2 98 1 10 8 9 9 2 99 1 9 6 7 2 9 100 4 3 9 3 5 1 101 5 11 10 14 12 1 102 7 12 4 13 3 2 103 5 5 4 4 12 9 104 2 13 13 11 13 13 105 10 2 5 4 12 7 106 11 7 4 11 6 4 107 12 12 1 9 11 11 108 12 9 4 14 12 6 109 12 7 13 2 9 11 110 9 11 3 4 1 3 111 10 5 12 11 4 4 112 4 13 7 12 1 5 113 9 13 10 11 11 6 114 10 14 14 10 1 3 115 2 14 1 10 4 5 116 10 12 12 7 11 10 117 9 11 2 12 8 11 118 2 8 5 2 12 14 119 1 8 13 3 7 8 120 9 4 7 5 4 2 121 13 2 12 7 1 12 122 11 10 9 7 5 11 123 8 12 2 2 12 7 124 5 2 14 3 4 13 125 1 8 8 1 5 9 126 14 5 11 10 13 3 127 14 1 4 13 2 4 128 4 4 5 11 3 10 129 10 9 2 3 3 11 130 11 4 8 14 3 4 131 5 1 14 8 11 2 132 14 3 11 6 12 5 133 13 4 4 1 10 1 134 6 10 11 6 5 1 135 5 8 12 5 1 7 136 4 5 9 6 9 2 137 13 2 4 4 2 3 138 11 2 2 5 9 3 139 8 1 10 12 2 8 140 12 7 9 11 4 1 141 12 1 4 14 3 13 142 11 2 7 10 4 1 143 3 4 12 11 11 11 144 3 3 4 2 12 11 145 1 5 9 4 2 1 146 6 1 12 2 10 5 147 10 5 1 12 2 14 148 2 11 7 9 4 11 149 7 4 4 5 14 12 150 12 5 2 1 10 12 151 5 9 2 11 6 1 152 12 14 3 6 1 14 153 5 9 11 10 1 4 154 2 5 12 14 10 10 155 4 5 8 4 5 6 156 10 12 4 6 12 5 157 4 2 1 13 6 8 158 9 10 10 14 5 3 159 6 14 10 11 3 3 160 2 9 10 12 5 7 161 13 3 7 10 5 12 162 6 4 1 2 5 13 163 6 1 13 4 14 13 164 2 12 1 14 1 9 165 4 11 13 2 6 10 166 1 10 7 4 5 8 167 7 2 2 10 13 4 168 8 2 11 4 6 14 169 4 8 2 6 2 3 170 7 1 12 11 2 9 171 5 6 10 4 13 4 172 5 10 4 11 9 3 173 3 11 9 3 2 3 174 8 15 6 20 17 19 175 21 10 15 3 7 11 176 11 7 17 20 14 9 177 16 6 17 13 21 21 178 10 15 22 6 17 21 179 15 7 17 10 22 22 180 3 20 8 15 20 16 181 17 21 10 16 6 22 182 6 21 14 14 14 16 183 7 17 3 20 10 7 184 16 19 14 17 7 21 185 20 16 7 15 22 10 186 20 10 18 11 22 18 187 18 7 19 15 7 22 188 21 18 7 21 16 3 189 14 13 7 22 17 13 190 19 7 8 12 10 17 191 15 3 21 14 9 7 192 19 6 15 7 14 14 193 4 17 10 15 20 19 194 21 6 18 4 20 16 195 2 19 8 17 6 13 196 12 12 6 17 4 20 197 16 21 12 10 19 16 198 14 14 15 2 7 21 199 8 16 21 6 22 16 200 14 17 22 14 17 20 201 10 21 7 15 21 18 202 16 13 20 18 21 12 203 15 7 4 22 14 13 204 7 19 14 8 15 4 205 4 5 3 20 7 16 206 22 18 6 18 13 20 207 19 6 16 3 13 3 208 18 6 22 7 20 18 209 10 17 11 21 8 13 210 7 10 17 19 10 14 Here, each of the numerals 1 to 22 (numeric identifiers) represents a 4mer and the pattern of numerals 1 to 10 of the sequences in the above list corresponds to the pattern of tetrameric oligonucleotide segments present in the oligonucleotides of Table I, which oligonucleotides have been found to be non-cross-hybridizing, as described further in the detailed examples. Each 4mer is selected from the group of 4mers consisting of WWWW, WWWX, WWWY, WWXW, WWXX, WWXY, WWYW, WWYX, WWYY, WXWW, WXWX, WXWY, WXXW, WXXX, WXXY, WXYW, WXYX, WXYY, WYWW, WYWX, WYWY, WYXW, WYXX, WYXY, WYYW, WYYX, WYYY, XWWW, XWWX, XWWY, XWXW, XWXX, XWXY, XWYW, XWYX, XWYY, XXWW, XXWX, XXWY, XXXW, XXXX, XXXY, XXYW, XXYX, XXYY, XYWW, XYWX, XYWY, XYXW, XYXX, XYXY, XYYW, XYYX, XYYY, YWWW, YWWX, YWWY, YWXW, YWXX, YWXY, YWYW, YWYX, YWYY, YXWW, YXWX, YXWY, YXXW, YXXX, YXXY, YXYW, YXYX, YXYY, YYWW, YYWX, YYWY, YYXW, YYXX, YYXY, YYYW, YYYX, and YYYY. Here W, X and Y represent nucleotide bases, A, G, C, etc., the assignment of bases being made according to rules described below.

Given this numeric pattern, a 4mer is assigned to a numeral. For example, 1=WXYY, 2=YWXY, etc. Once a given 4mer has been assigned to a given numeral, it is not assigned for use in the position of a different numeral. It is possible, however, to assign a different 4mer to the same numeral. That is, for example, the numeral 1 in one position could be assigned WXYY and another numeral 1, in a different position, could be assigned XXXW, but none of the other numerals 2 to 10 can then be assigned WXYY or XXXW. A different way of saying this is that each of 1 to 10 is assigned a 4mer from the list of eighty-one 4mers indicated so as to be different from all of the others of 1 to 10.

In the case of the specific oligonucleotides given in Table I, 1=WXYY, 2=YWXY, 3=XXXW, 4=YWYX, 5=WYXY, 6=YYWX, 7=YWXX, 8=WYXX, 9=XYYW, 10=XYWX, 11=YYXW, 12=WYYX, 13=XYXW, 14=WYYY, 15=WXYW, 16=WYXW, 17=WXXW, 18=WYYW, 19=XYYX, 20=YXYX, 21=YXXY and 22=XYXY.

Once the 4mers are assigned to positions according to the above pattern, a particular set of oligonucleotides can be created by appropriate assignment of bases, A, T/U, G, C to W, X, Y. These assignments are made according to one of the following two sets of rules:

-   (i) Each of W, X and Y is a base in which:     -   (a) W=one of A, T/U, G, and C,         -   X=one of A, T/U, G, and C,         -   Y=one of A, T/U, G, and C,         -   and each of W, X and Y is selected so as to be different             from all of the others of W, X and Y,     -   (b) an unselected said base of (i)(a) can be substituted any         number of times for any one of W, X and Y.         or -   (ii) Each of W, X and Y is a base in which:     -   (a) W=G or C,         -   X=A or T/U,         -   Y=A or T/U,         -   and X≠Y, and     -   (b) a base not selected in (ii)(a) can be inserted into each         sequence at one or more locations, the location of each         insertion being the same in each sequence as that of every o         sequence of the set;

In the case of the specific oligonucleotides given in Table I, W=G, X=A and Y=T.

In any case, given a set of oligonucleotides generated according to one of these sets of rules, it is possible to modify the members of a given set in relatively minor ways and thereby obtain a different set of sequences while more or less maintaining the cross-hybridization properties of the set subject to such modification. In particular, it is possible to insert up to 3 of A, T/U, G and C at any location of any sequence of the set of sequences. Alternatively, or additionally, up to 3 bases can be deleted from any sequence of the set of sequences.

A person skilled in the art would understand that given a set of oligonucleotides having a set of properties making it suitable for use as a family of tags (or tag complements) one can obtain another family with the same property by reversing the order of all of the members of the set. In other words, all the members can be taken to be read 51 to 31 or to be read 3′ to 5′.

A family of complements of the present invention is based on a given set of oligonucleotides defined as described above. Each complement of the family is based on a different oligonucleotide of the set and each complement contains at least 10 consecutive (i.e., contiguous) bases of the oligonucleotide on which it is based. For a given family of complements where one is seeking to reduce or minimize inter-sequence similarity that would result in cross-hybridization, each and every pair of complements meets particular homology requirements. Particularly, subject to limited exceptions, described below, any two complements within a set of complements are generally required to have a defined amount of dissimilarity.

In order to notionally understand these requirements for dissimilarity as they exist for a given pair of complements of a family, a phantom sequence is generated from the pair of complements. A “phantom” sequence is a single sequence that is generated from a pair of complements by selection, from each complement of the pair, of a string of bases wherein the bases of the string occur in the same order in both complements. An object of creating such a phantom sequence is to create a convenient and objective means of comparing the sequence identity of the two parent sequences from which the phantom sequence is created.

A phantom sequence may thus be generated from exemplary Sequence 1 and Sequence 2 as follows: Sequence 1: ATGTTTAGTGAAAAGTTAGTATTG    *        • Sequence 2: ATGTTAGTGAATAGTATAGTATTG            •   ♦ Phantom Sequence: ATGTTAGTGAAAGTTAGTATTG

The phantom sequence generated from these two sequences is thus 22 bases in length. That is, one can see that there are 22 identical bases with identical sequence (the same order) in Sequence Nos. 1 and 2. There is a total of three insertions/deletions and mismatches present in the phantom sequence when compared with the sequences from which it was generated:

-   ATGT-TAGTGAA-AGT-TAGTATTG     The dashed lines in this latter representation of the phantom     sequence indicate the locations of the insertions/deletions and     mismatches in the phantom sequence relative to the parent sequences     from which it was derived. Thus, the “T” marked with an asterisk in     Sequence 1, the “A” marked with a diamond in Sequence 2 and the     “A-T” mismatch of Sequences 1 and 2 marked with two dots were     deleted in generating the phantom sequence.

A person skilled in the art will appreciate that the term “insertion/deletion” is intended to cover the situations indicated by the asterisk and diamond. Whether the change is considered, strictly speaking, an insertion or deletion is merely one of vantage point. That is, one can see that the fourth base of Sequence 1 can be deleted therefrom to obtain the phantom sequence, or a “T” can be inserted after the third base of the phantom sequence to obtain Sequence 1.

One can thus see that if it were possible to create a phantom sequence by elimination of a single insertion/deletion from one of the parent sequences, that the two parent sequences would have identical homology over the length of the phantom sequence except for the presence of a single base in one of the two sequences being compared. Likewise, one can see that if it were possible to create a phantom sequence through deletion of a mismatched pair of bases, one base in each parent, that the two parent sequences would have identical homology over the length of the phantom sequence except for the presence of a single base in each of the sequences being compared. For this reason, the effect of an insertion/deletion is considered equivalent to the effect of a mismatched pair of bases when comparing the homology of two sequences.

Once a phantom sequence is generated, the compatibility of the pair of complements from which it was generated within a family of complements can be systematically evaluated:

According to one embodiment of the invention, a pair of complements is compatible for inclusion within a family of complements if any phantom sequence generated from the pair of complements has the following properties:

-   -   Any consecutive sequence of bases in the phantom sequence which         is identical to a consecutive sequence of bases in each of the         first and second complements from which it is generated is no         more ((¾×L)−1) bases in length;     -   The phantom sequence, if greater than or equal to (⅚×L) in         length, contains at least 3 insertions/deletions or mismatches         when compared to t first and second complements from which it is         generated; and     -   The phantom sequence is not greater than or equal to ( 11/12×L)         in length.

Here, L₁ is the length of the first complement, L₂ is the length of the second complement, and L=L₁, or if L₁≠L₂, L is the greater of L₁ and L₂.

In particular preferred embodiments of the invention, all pairs of complements of a given set have the properties set out above. Under particular circumstances, it may be advantageous to have a limited number of complements that do not meet all of these requirements when compared to every other complement in a family.

In one case, for any first complement there are at most two second complements in the family which do not meet all of the three listed requirements. For two such complements, there would thus be a greater chance of cross-hybridization between their tag counterparts and the first complement. In another case, for any first complement there is at most one second complement which does not meet all of three listed requirements.

It is also possible, given this invention, to design a family of complements where a specific number or specific portion of the complements do not meet the three listed requirements. For example, a set could be designed where only one pair of complements within the set do not meet the requirements when compared to each other. There could be two pairs, three pairs, and any number of pairs up to and including all possible pairs. Alternatively, it may be advantageous to have a given proportion of pairs of complements that do not meet the requirements, say 10% of pairs, when compared with other sequences that do not meet one or more of the three requirements listed. This number could instead by 5%, 15%, 20%, 25%, 30%, 35%, or 40%.

The foregoing comparisons would generally be largely carried out using appropriate computer software. Although notionally described in terms of a phantom sequence for the sake of clarity and understanding, it will be understood that a competent computer programmer can carry out pairwise comparisons of complements in any number of ways using logical steps that obtain equivalent results.

The symbols A, G, T/U; C take on their usual meaning in the art here. In the case of T and U, a person skilled in the art would understand that these are equivalent to each other with respect to the inter-strand hydrogen-bond (Watson-Crick) binding properties at work in the context of this invention. The two bases are thus interchangeable and hence the designation of T/U.

Analogues of the naturally occurring bases can be inserted in their respective places where desired. Analogues can be defined as any non-natural base, such as peptide nucleic acids and the like.

Other aspects of the invention are described below, particularly numbered paragraphs at the end of this specification.

In another broad embodiment A family of 1168 sequences was obtained using a computer algorithm to have desirable hybridization properties for use in nucleic acid detection assays. The sequence set of 1168 oligonucleotides was partially characterized in hybridization assays, demonstrating the ability of family members to correctly hybridize to their complementary sequences with minimal cross hybridization. These are the sequences having SEQ ID NOs:1 to 1168 of Table II.

Variant families of sequences (seen as tags or tag complements) of a family of sequences taken from Table II are also part of the invention. For the purposes of discussion, a family or set of oligonucleotides will-often be described as a family of tag complements, but it will be understood that such a set could just easily be a family of tags.

A family of complements is obtained from a set of oligonucleotides based on a family of oligonucleotides such as those of Table II. To simplify discussion, providing a family of complements based on the oligonucleotides of Table II will be described.

Firstly, the groups of sequences based on the oligonucleotides of Table II can be represented as shown in Table IIA. TABLE IIA Numeric sequences corresponding to nucleotide patterns of a set of oligonucleotides Sequence Numeric Pattern Identifier 1 1 1 2 2 3 2 3 1 1 1 3 1 2 2 3 2 2 2 3 2 3 2 1 1 3 2 2 1 3 1 3 2 2 1 1 2 2 3 2 1 2 2 2 3 1 2 3 1 2 1 2 3 2 2 1 1 1 3 2 1 1 3 2 3 2 2 3 1 1 1 2 3 2 3 2 3 1 2 3 2 2 1 3 1 1 3 2 1 2 1 2 2 3 2 3 1 1 2 4 2 2 2 3 2 3 2 1 3 1 1 2 1 2 3 2 3 2 2 3 2 2 1 1 5 1 2 1 1 3 2 3 2 1 1 3 2 3 1 1 1 2 1 1 3 1 1 3 1 6 1 1 3 1 3 2 1 2 2 2 3 2 2 3 2 3 1 3 2 2 1 1 1 2 7 3 2 3 2 2 2 1 2 3 2 2 1 2 1 2 3 2 3 1 1 3 2 2 2 8 1 1 1 3 1 3 1 1 2 1 3 1 1 2 1 2 3 2 3 2 1 1 3 2 9 2 1 2 3 1 1 1 3 1 3 2 3 1 3 1 2 1 1 2 3 2 2 2 1 10 1 2 3 1 3 1 1 1 2 1 2 3 2 2 1 3 1 1 2 3 2 3 1 2 11 2 2 1 3 2 2 3 2 2 3 1 2 3 2 2 2 1 3 2 1 3 2 2 2 12 3 2 1 1 1 3 1 3 2 1 2 1 1 3 2 2 2 3 1 2 3 1 2 1 13 1 1 1 3 2 1 1 3 1 1 2 3 1 2 3 2 1 1 2 1 1 3 2 3 14 3 2 1 3 1 1 1 2 1 3 2 2 2 1 2 2 3 1 2 3 1 2 2 3 15 2 3 2 1 1 3 2 3 1 1 1 2 1 3 2 3 1 3 2 2 1 2 2 2 16 1 1 1 2 1 3 1 2 3 1 2 1 2 1 1 3 2 3 1 3 1 1 2 3 17 1 2 1 1 3 2 2 1 2 1 1 3 2 3 2 2 1 2 3 2 3 1 3 2 18 2 1 2 1 3 1 2 1 1 1 3 1 3 1 2 3 1 2 2 2 3 2 2 3 19 1 3 1 3 2 2 3 1 3 1 1 2 3 2 1 2 1 3 2 1 2 2 1 2 20 1 1 3 2 1 3 2 2 2 3 2 1 1 3 1 1 2 3 1 2 2 3 2 1 21 2 2 1 2 3 1 1 1 2 2 3 1 3 2 3 1 1 3 1 2 2 3 1 2 22 3 2 1 2 1 2 3 2 1 1 1 2 2 3 2 2 1 2 3 2 2 3 1 3 23 3 1 1 2 2 3 2 1 2 1 1 1 3 2 1 2 2 1 3 1 2 3 2 3 24 2 1 3 1 2 3 1 3 1 2 2 1 1 3 2 3 2 2 1 2 2 2 3 1 25 3 2 2 1 1 3 2 2 2 3 2 2 2 1 2 3 2 1 2 1 3 1 1 3 26 3 1 3 2 1 2 2 1 3 2 1 1 1 3 2 3 1 2 1 2 3 1 2 1 27 3 2 3 1 1 2 3 1 2 2 2 1 3 2 1 1 1 2 3 1 2 2 3 1 28 3 1 2 2 3 1 1 3 2 2 1 2 1 3 1 1 1 2 3 1 2 2 1 3 29 1 3 2 3 1 2 1 1 1 2 3 2 2 1 3 2 2 3 1 1 2 2 3 2 30 2 1 2 1 2 1 3 2 1 1 1 2 3 2 2 2 3 2 3 2 3 2 2 3 31 2 2 1 1 3 2 3 2 2 1 3 2 2 1 2 2 2 3 2 2 3 2 1 3 32 3 2 1 3 2 1 1 2 1 2 3 1 1 3 2 3 1 3 1 1 2 1 2 1 33 2 1 3 2 3 2 1 2 1 3 1 1 2 3 2 1 3 1 2 2 2 1 3 2 34 2 2 3 2 1 3 1 2 2 1 3 1 2 3 2 3 2 2 2 3 2 1 1 1 35 2 1 3 2 1 2 1 3 1 3 2 1 3 1 3 1 2 3 1 2 1 2 2 2 36 1 2 2 3 2 3 1 1 1 3 1 1 1 3 1 3 1 1 3 1 1 1 2 2 37 2 3 2 3 1 3 1 1 2 2 1 1 3 1 2 2 1 1 3 1 1 2 3 2 38 1 2 1 2 2 1 3 2 2 1 1 3 1 1 1 3 1 1 3 1 3 2 2 3 39 2 2 3 2 1 3 2 2 3 1 3 1 1 1 2 1 2 3 2 1 3 2 2 2 40 2 1 3 1 3 2 2 3 2 2 1 1 1 3 1 3 2 3 2 1 1 1 2 1 41 3 2 2 1 2 3 1 2 3 2 3 2 1 2 1 1 3 2 1 1 2 1 2 3 42 2 2 2 3 2 2 1 3 1 1 2 3 1 3 1 1 3 1 2 2 2 1 2 3 43 1 3 2 1 2 1 3 2 2 2 1 1 1 3 1 1 3 2 1 3 2 1 3 1 44 3 2 3 1 3 1 2 1 2 1 3 1 2 2 2 1 3 1 1 1 3 2 1 1 45 2 2 3 2 2 2 1 2 1 3 2 3 1 1 3 2 3 1 1 2 1 3 2 1 46 1 1 3 2 1 1 3 2 1 3 2 1 1 2 1 3 2 3 2 3 2 2 1 1 47 1 2 2 2 3 2 3 1 3 2 2 1 2 3 1 1 1 3 1 2 1 1 3 1 48 3 1 1 1 3 2 1 3 1 3 1 1 2 1 1 1 3 1 2 1 1 3 1 1 49 1 2 2 2 1 1 3 1 2 2 3 2 2 1 1 3 1 3 2 1 3 1 1 3 50 3 2 2 2 1 1 1 3 1 2 2 3 2 1 1 3 1 1 2 3 2 3 2 1 51 2 2 2 3 2 3 1 1 3 1 2 3 1 1 3 2 1 2 2 2 3 2 1 2 52 2 3 2 3 2 2 2 1 3 1 1 2 2 2 1 3 2 1 2 3 2 3 2 1 53 3 1 2 1 1 2 3 1 2 2 1 2 1 3 1 1 1 3 2 3 2 2 2 3 54 3 2 2 1 2 2 2 3 2 1 1 3 2 2 1 1 3 1 2 1 3 2 1 3 55 1 3 2 2 2 1 2 2 3 1 1 1 3 1 3 2 2 2 3 1 1 2 1 3 56 2 2 3 2 3 2 2 2 1 2 2 3 2 3 2 1 3 2 2 2 1 1 1 3 57 1 2 2 3 2 3 1 3 1 1 3 1 2 1 2 3 1 1 1 3 2 2 1 2 58 2 3 1 3 1 1 2 3 2 1 1 1 3 1 1 2 3 2 2 2 1 2 2 3 59 1 2 3 2 3 1 1 1 3 2 2 1 2 3 1 2 3 2 2 1 1 2 2 3 60 3 2 2 2 1 3 2 1 2 2 1 3 2 2 3 2 2 1 1 3 1 2 2 3 61 3 1 2 2 3 1 2 1 2 2 2 3 1 1 2 3 2 2 2 3 2 2 2 3 62 2 3 1 1 2 2 3 1 1 1 3 2 3 2 1 1 2 3 2 2 3 2 1 2 63 3 1 2 2 3 2 1 2 2 3 2 2 3 1 3 1 1 2 1 3 1 1 2 1 64 1 1 1 2 2 2 3 1 3 1 2 2 2 3 2 3 1 2 1 3 1 3 2 1 65 3 2 1 1 2 2 1 3 1 2 2 2 3 2 2 2 3 2 2 3 2 2 3 2 66 3 2 2 2 3 2 1 2 2 3 2 2 1 3 2 3 1 1 2 1 2 1 3 2 67 1 2 3 2 1 3 2 1 3 2 1 3 1 2 3 2 2 2 1 2 3 1 1 2 68 2 3 2 2 2 1 1 1 3 1 2 3 1 2 2 3 1 1 3 1 1 1 2 3 69 2 3 2 3 1 2 1 1 2 3 1 2 3 2 2 1 2 2 2 3 2 3 2 1 70 1 2 1 3 2 2 3 2 3 1 3 1 1 2 2 2 3 2 1 1 2 2 1 3 71 1 2 1 3 1 2 3 2 1 1 3 1 3 1 1 1 2 2 3 2 3 1 1 1 72 1 3 1 2 2 1 1 3 1 3 1 1 3 2 2 1 1 2 1 3 1 3 2 1 73 3 1 1 3 2 1 1 1 2 2 3 2 3 1 1 2 3 1 1 1 3 1 1 1 74 1 1 2 3 2 1 1 3 1 1 1 3 1 1 3 1 2 2 3 2 2 3 2 1 75 2 2 2 3 1 2 2 2 1 2 3 2 3 2 2 1 2 3 2 2 3 1 3 2 76 3 2 1 2 2 3 1 3 1 1 1 2 2 2 3 1 1 3 1 1 2 3 1 1 77 3 1 1 2 2 3 2 1 2 3 1 1 1 2 3 1 1 2 2 3 2 1 1 3 78 2 1 2 2 3 2 1 3 1 1 3 2 1 1 1 3 2 2 1 3 1 1 3 2 79 2 2 2 1 2 3 2 1 1 2 3 1 2 1 1 3 2 3 2 1 3 2 2 3 80 1 2 1 2 1 3 2 2 3 1 1 1 2 2 3 2 3 1 2 1 3 2 3 2 81 1 2 1 1 3 1 1 1 2 2 1 3 1 3 1 3 2 2 3 2 1 1 1 3 82 3 1 1 2 2 3 2 3 1 1 1 2 3 2 3 1 2 2 3 1 2 1 2 1 83 1 1 1 2 1 1 3 2 1 3 2 2 2 1 1 2 3 1 3 1 3 1 1 3 84 3 1 2 2 1 1 1 3 1 1 3 2 1 1 3 2 3 1 1 2 3 2 2 2 85 2 1 2 3 2 3 2 3 2 2 3 2 2 2 1 3 2 3 2 2 1 2 2 1 86 3 1 3 2 2 1 2 1 2 3 2 1 3 2 2 1 3 1 3 2 2 1 2 1 87 3 1 1 1 3 1 1 1 3 1 1 3 2 3 2 2 1 1 3 2 2 1 1 1 88 2 1 3 2 1 2 2 1 3 2 1 1 3 2 1 2 3 2 3 1 2 2 3 2 89 2 2 3 2 3 2 3 1 2 2 3 1 1 2 1 2 2 3 2 3 1 1 1 2 90 1 2 3 2 3 1 1 1 3 1 3 2 2 1 1 3 2 3 1 2 2 1 1 1 91 3 1 2 2 3 1 1 2 3 1 2 2 3 1 3 1 2 1 2 3 2 1 1 1 92 1 1 3 1 2 3 1 2 1 3 2 2 1 1 3 2 3 2 1 1 3 2 2 1 93 2 1 3 2 2 3 2 2 1 2 2 3 1 3 1 1 2 2 2 1 3 1 1 3 94 2 2 2 1 2 1 3 2 3 1 1 2 2 1 2 3 1 3 2 3 1 1 1 3 95 3 1 2 1 3 1 2 2 2 1 3 1 1 2 3 1 1 2 2 1 1 3 2 3 96 2 2 2 3 1 1 3 1 1 3 1 3 1 2 2 2 3 1 1 1 2 2 3 1 97 1 2 3 1 1 2 1 1 3 1 3 2 2 3 1 2 1 1 1 2 3 2 3 1 98 2 3 2 2 2 1 2 3 2 1 3 2 3 2 1 3 1 2 2 3 1 1 2 2 99 2 2 2 1 1 3 2 3 1 3 2 2 1 2 1 3 1 1 3 2 1 3 2 1 100 3 1 2 2 2 1 2 3 2 3 2 2 2 3 1 1 3 2 2 1 1 3 1 2 101 2 1 3 2 2 1 3 1 3 1 1 1 3 2 3 1 2 1 1 1 3 2 2 1 102 3 2 1 1 2 3 1 2 1 1 2 3 1 1 3 2 3 2 1 2 1 2 1 3 103 1 1 2 3 1 1 3 2 3 2 2 1 3 2 1 2 1 3 1 2 1 3 2 1 104 2 1 1 1 2 2 3 1 3 2 2 2 3 2 2 2 3 1 2 2 3 2 1 3 105 2 1 1 2 3 1 1 3 1 1 2 1 1 3 2 1 2 3 1 3 2 3 2 2 106 1 1 1 2 3 2 1 1 2 1 3 2 3 2 2 3 2 2 1 3 2 2 1 3 107 1 3 1 3 2 2 1 3 2 3 1 1 1 2 3 2 2 3 2 2 1 1 1 2 108 3 1 1 1 2 1 3 1 1 1 2 3 2 1 2 2 3 2 2 2 3 2 3 1 109 1 3 2 2 1 2 1 1 3 2 2 2 3 2 3 1 3 1 1 2 2 1 1 3 110 3 1 3 2 2 2 1 2 1 3 2 2 1 3 1 1 2 1 2 3 2 2 3 2 111 1 3 1 3 2 2 1 2 2 1 3 1 1 3 1 1 3 1 2 2 2 1 1 3 112 3 1 3 2 2 1 1 2 3 1 1 1 2 1 1 3 2 1 2 2 2 3 2 3 113 1 2 3 1 2 3 1 1 2 1 3 2 2 3 1 1 3 2 1 2 1 2 1 3 114 1 2 1 3 1 2 1 2 3 1 3 1 2 3 1 1 1 3 2 2 1 3 2 1 115 2 1 2 3 2 1 1 1 3 1 1 1 3 2 3 1 1 1 3 1 1 3 1 1 116 2 3 1 1 2 3 2 1 3 1 1 1 2 3 1 1 2 3 2 2 3 1 1 1 117 1 1 2 2 3 1 1 2 1 3 2 3 2 3 2 3 1 3 2 2 2 1 1 2 118 1 3 1 2 1 2 2 3 2 2 2 3 1 2 2 1 1 2 3 1 1 3 1 3 119 1 1 1 3 2 2 3 2 1 1 1 3 2 2 3 1 1 3 1 2 1 1 1 3 120 3 2 2 1 1 3 1 3 1 2 2 1 2 3 1 3 1 2 3 2 1 2 2 1 121 1 3 1 1 3 1 2 1 2 1 1 3 1 1 3 1 2 2 3 1 1 2 2 3 122 3 2 1 3 1 1 1 2 2 2 3 1 1 2 2 3 1 2 3 2 3 1 1 1 123 1 1 3 1 3 2 1 3 1 2 2 3 1 2 1 1 3 2 1 2 1 2 3 1 124 2 3 1 2 1 2 1 3 2 1 3 2 3 1 1 3 1 1 1 2 1 1 3 2 125 1 3 1 2 1 1 2 3 1 2 3 1 3 1 1 1 2 3 1 1 3 1 2 1 126 1 2 3 2 3 1 1 1 3 2 1 2 2 2 3 2 3 1 2 1 2 1 3 2 127 1 1 2 1 1 3 1 3 1 1 2 2 3 1 2 1 2 3 1 1 3 1 2 3 128 2 1 1 3 2 3 2 1 2 2 2 1 3 2 1 3 1 1 2 3 1 1 3 2 129 2 1 2 3 2 2 1 3 1 2 2 2 3 2 2 3 1 3 1 2 2 3 1 2 130 1 3 2 2 2 3 2 1 2 3 1 1 3 1 3 1 2 1 3 2 1 2 2 2 131 3 1 3 1 1 1 2 3 2 2 1 2 3 2 1 2 2 2 1 3 2 1 3 2 132 2 1 2 3 2 3 1 3 1 1 2 3 2 3 2 2 2 3 1 2 2 2 1 1 133 3 2 1 2 3 2 2 2 3 2 2 2 1 2 1 3 1 1 2 3 2 1 2 3 134 3 1 3 2 1 2 1 2 1 3 1 1 3 1 1 1 3 1 1 1 2 2 2 3 135 1 2 3 1 3 2 3 1 1 3 2 1 1 1 2 3 2 1 3 2 2 1 2 2 136 2 2 1 1 3 1 1 3 2 3 1 3 2 2 1 2 2 3 2 3 1 2 1 2 137 1 2 3 1 1 1 2 3 1 3 1 1 2 1 2 2 3 2 2 3 2 2 2 3 138 3 1 2 2 1 1 2 3 1 2 2 1 2 3 2 3 1 1 2 2 3 1 2 3 139 3 1 1 1 2 3 2 2 1 1 1 3 1 2 1 2 3 1 1 1 3 2 1 3 140 2 1 2 2 3 2 2 3 1 2 2 2 3 1 2 1 2 2 1 3 2 3 2 3 141 2 2 2 1 2 3 2 2 2 3 2 3 2 1 2 3 2 1 1 3 2 1 3 2 142 1 1 2 2 3 1 1 1 3 1 1 2 2 3 2 3 2 3 1 1 2 2 3 1 143 2 3 1 3 2 2 2 3 1 1 2 2 2 3 2 2 2 3 1 3 2 1 1 2 144 3 1 2 3 2 1 2 1 1 2 3 1 2 3 2 3 2 3 2 1 1 1 2 2 145 1 2 3 2 3 1 3 1 3 1 1 3 1 1 2 2 2 3 2 2 2 1 2 2 146 3 2 3 1 2 1 1 1 3 2 1 2 2 3 2 2 3 1 2 1 3 1 1 1 147 3 1 1 3 2 1 3 1 1 2 1 3 1 1 1 3 2 2 1 1 2 1 3 1 148 2 2 3 2 3 2 1 3 2 2 1 1 3 1 3 2 2 3 2 2 2 1 1 2 149 2 1 3 2 1 3 2 1 1 3 2 2 3 2 2 1 3 1 1 2 1 3 2 2 150 1 1 2 2 2 3 1 1 3 2 1 2 1 1 2 3 1 1 2 3 2 3 2 3 151 2 1 3 1 1 1 2 2 3 2 1 3 2 1 2 2 2 3 1 3 1 3 1 1 152 2 3 2 1 2 1 2 3 2 2 1 1 2 3 1 3 1 2 3 2 2 3 2 1 153 2 1 2 2 2 3 1 2 1 1 3 1 3 1 1 2 3 1 1 3 1 1 3 2 154 2 2 3 1 1 2 1 3 2 3 2 1 1 2 3 1 1 2 1 2 3 1 2 3 155 3 2 1 3 2 2 2 3 2 3 1 1 2 1 3 1 1 2 2 1 3 2 2 2 156 1 1 1 3 1 2 3 1 2 2 3 2 1 1 2 2 2 3 2 3 2 3 1 1 157 3 1 1 3 1 2 2 3 2 2 3 1 3 2 2 1 1 2 1 3 1 2 1 1 158 1 3 1 2 2 1 2 3 2 1 3 2 3 1 2 3 2 1 1 1 2 3 2 2 159 3 1 1 2 2 2 1 3 1 2 3 2 1 3 1 2 1 2 3 1 1 2 3 2 160 3 1 2 1 3 1 1 3 2 3 2 1 2 2 1 1 3 2 1 1 3 2 2 1 161 2 1 2 3 1 1 2 2 1 2 3 1 3 1 1 3 1 1 2 1 3 1 3 2 162 2 2 2 3 2 2 1 2 3 1 1 3 2 3 1 2 2 2 3 2 2 2 3 2 163 3 2 1 1 1 3 1 2 2 3 2 3 2 2 1 2 1 2 3 1 1 1 2 3 164 2 2 3 2 3 1 2 1 3 2 1 3 2 2 1 3 1 2 1 2 2 2 3 2 165 3 1 1 2 2 1 1 3 1 2 1 1 1 3 1 1 3 1 3 1 1 3 2 1 166 3 1 2 2 3 2 1 3 1 1 2 3 1 1 2 2 2 3 2 1 3 2 1 2 167 1 1 1 2 1 1 3 1 3 1 3 1 3 1 1 2 3 1 2 2 2 1 3 2 168 1 1 2 2 1 2 3 2 3 1 1 2 1 3 1 2 2 3 2 2 3 1 1 3 169 2 2 1 1 3 1 2 2 2 1 2 3 2 3 1 2 1 3 2 1 3 1 3 2 170 2 2 1 1 1 3 1 2 1 3 2 3 2 2 2 3 2 2 3 2 3 2 2 1 171 2 1 2 2 3 1 2 2 2 1 2 3 1 1 3 1 3 2 1 2 1 3 2 3 172 1 1 1 2 2 2 3 1 2 3 1 3 2 1 3 2 2 2 1 1 3 1 3 1 173 1 2 1 1 1 3 2 2 3 2 2 2 3 1 2 3 2 2 2 3 1 1 2 3 174 3 1 2 2 3 2 3 1 2 3 1 1 2 1 1 2 3 2 2 1 2 2 3 1 175 3 1 2 3 1 1 3 1 1 1 2 1 2 3 1 2 1 2 3 1 1 2 1 3 176 2 2 1 1 1 3 2 2 1 2 2 3 1 1 3 2 3 1 1 3 2 2 3 1 177 2 2 3 2 1 1 3 1 1 1 2 1 3 1 3 1 2 2 2 3 2 3 2 2 178 3 1 3 1 2 2 3 1 3 2 2 2 1 1 3 2 1 2 2 1 3 1 2 2 179 1 3 2 3 1 2 1 1 2 1 3 1 1 2 3 1 2 1 1 1 2 3 2 3 180 3 1 2 1 1 2 1 3 2 3 1 1 2 2 2 3 1 3 2 2 3 2 1 2 181 1 3 1 2 1 2 2 2 3 2 1 3 2 1 3 1 1 1 3 2 1 2 3 2 182 3 2 2 1 2 3 1 1 2 3 2 2 3 1 1 2 2 2 3 1 1 2 3 2 183 1 2 3 1 1 1 3 1 2 2 2 1 3 2 2 3 2 3 1 3 1 2 1 2 184 1 1 1 2 1 3 1 3 1 1 3 2 2 1 2 3 1 2 3 2 3 1 2 1 185 2 2 1 3 2 3 1 3 1 1 1 2 3 2 2 2 1 1 2 3 2 3 1 2 186 2 3 1 1 3 1 1 2 1 2 3 2 3 1 1 1 2 2 1 3 2 2 2 3 187 3 2 2 2 3 1 2 1 3 2 2 2 1 1 2 3 1 3 2 1 2 2 3 1 188 3 2 2 3 2 1 1 3 2 1 1 2 3 1 2 1 1 1 3 2 1 2 3 1 189 2 1 1 3 1 3 2 1 3 2 1 1 2 2 3 2 2 3 2 2 2 1 3 1 190 2 2 2 3 1 3 1 3 1 3 2 1 2 3 2 1 2 3 1 2 2 1 2 2 191 1 2 2 3 1 2 2 3 2 3 1 1 2 2 1 3 1 2 1 3 1 1 3 1 192 3 1 2 2 1 3 2 1 2 2 2 1 3 2 1 3 2 1 1 2 1 3 1 3 193 2 1 2 3 2 1 2 2 1 3 1 3 1 2 1 2 2 3 1 1 1 3 2 3 194 2 1 2 3 2 3 1 1 1 3 2 1 1 2 3 1 2 1 1 1 2 3 1 3 195 3 2 1 1 2 2 1 3 2 1 1 2 3 1 2 2 2 3 1 1 2 3 1 3 196 3 2 2 2 1 2 2 3 2 1 1 1 3 1 2 3 2 1 1 3 2 3 1 1 197 2 1 3 2 1 3 1 1 2 2 3 2 2 3 2 2 1 1 1 3 1 1 2 3 198 2 1 2 2 3 2 2 1 3 2 2 1 2 3 2 1 3 2 3 2 3 2 1 1 199 3 1 3 2 3 1 1 1 3 2 2 1 2 1 2 3 1 1 1 3 2 1 2 1 200 1 2 1 2 1 3 1 1 3 2 2 3 1 2 3 1 3 2 2 2 1 2 3 1 201 2 2 2 1 3 1 1 3 2 1 1 3 1 1 2 1 1 3 2 3 1 3 2 1 202 2 3 2 3 2 1 2 1 1 3 1 2 1 2 2 2 3 2 1 1 3 1 1 3 203 2 1 3 1 1 3 1 3 2 2 3 2 1 2 2 3 2 2 1 2 1 1 3 2 204 3 2 3 2 2 1 2 2 1 3 2 2 2 1 1 3 2 2 1 3 1 3 2 1 205 1 1 2 1 2 1 3 2 3 1 2 3 2 3 1 1 1 2 2 3 1 1 2 3 206 2 2 1 3 1 3 1 1 2 1 3 1 3 2 3 1 2 2 1 2 1 3 2 2 207 3 1 1 3 2 3 1 3 2 2 1 1 2 3 1 2 2 2 3 2 1 1 1 2 208 1 1 2 3 2 1 1 1 3 2 1 1 1 3 1 1 1 3 2 3 1 2 3 1 209 3 2 2 1 3 2 2 1 2 3 1 2 3 1 1 2 1 2 2 3 2 3 2 1 210 1 1 1 2 3 1 3 2 2 1 3 1 3 2 1 3 1 1 2 2 1 2 3 2 211 3 1 2 1 2 1 3 1 1 3 1 2 2 1 3 2 2 1 3 2 3 1 2 1 212 1 2 1 3 2 2 2 3 2 2 3 1 3 1 2 2 2 1 2 3 1 3 2 1 213 2 1 3 1 1 2 1 3 2 2 1 3 2 1 3 2 1 1 3 1 3 2 1 2 214 3 1 1 2 2 2 3 2 1 2 2 3 2 3 1 1 3 2 2 2 1 3 2 1 215 3 2 1 3 2 1 1 3 1 1 3 1 3 1 1 2 2 1 3 1 2 2 1 1 216 1 1 2 3 2 3 2 2 1 2 3 2 1 2 3 2 1 1 1 2 1 3 2 3 217 3 1 1 2 2 1 3 2 2 1 3 1 3 2 1 1 1 2 2 3 2 2 2 3 218 3 1 1 1 2 2 3 1 1 3 1 2 1 3 2 1 1 3 1 1 1 2 3 1 219 3 2 3 2 1 2 2 1 2 3 2 3 1 2 2 2 1 2 3 1 2 1 3 1 220 2 1 2 2 1 2 3 1 3 1 1 1 3 2 2 3 1 1 2 1 3 2 1 3 221 2 1 2 3 2 1 2 2 3 2 1 2 2 3 1 3 2 1 3 1 2 3 1 1 222 3 2 3 1 2 2 3 1 1 2 1 3 2 1 3 1 2 2 3 2 2 2 1 1 223 1 3 2 1 1 3 2 2 3 2 2 2 3 1 2 2 3 1 1 1 2 2 2 3 224 3 1 1 3 2 2 2 3 1 2 2 2 1 1 3 2 2 2 1 1 3 1 1 3 225 3 1 3 1 1 3 1 2 1 1 1 2 3 1 2 1 2 2 3 2 2 1 2 3 226 1 2 3 1 2 3 1 3 2 2 3 2 2 1 1 2 1 3 2 2 1 3 2 2 227 2 1 2 3 1 2 1 2 2 2 3 1 1 3 1 3 2 3 2 2 1 1 3 1 228 3 1 3 1 2 3 1 2 2 1 1 1 3 2 3 1 2 2 2 1 2 3 1 1 229 1 2 1 3 2 2 1 1 3 1 3 2 3 1 2 3 1 3 1 1 2 1 1 1 230 2 2 2 1 2 2 3 2 2 1 3 1 2 1 1 1 3 1 3 2 2 3 1 3 231 1 3 1 1 2 1 2 2 3 1 2 1 3 2 2 3 1 1 3 2 2 3 1 1 232 2 1 3 2 3 2 1 1 1 3 2 3 2 1 3 1 2 2 3 2 1 1 1 2 233 1 3 2 1 3 2 3 1 2 1 2 3 1 2 2 2 3 1 1 2 1 2 2 3 234 2 3 2 1 2 2 3 1 1 2 2 1 3 1 1 2 1 3 2 3 1 3 1 1 235 2 3 1 2 1 2 3 1 3 1 2 1 3 1 1 3 2 2 2 1 1 2 3 2 236 3 1 1 3 1 1 3 2 1 1 3 2 1 2 1 1 1 3 2 1 1 1 2 3 237 2 2 2 1 1 3 2 3 2 3 1 2 1 1 3 1 1 1 3 1 2 1 3 1 238 2 1 2 2 3 2 2 3 1 1 2 3 2 3 2 2 2 1 1 1 3 1 3 1 239 3 1 1 2 1 1 2 3 1 2 3 1 3 1 2 3 1 2 2 1 2 2 3 1 240 2 1 3 1 3 1 1 1 3 1 3 1 3 1 1 2 2 3 2 1 2 2 1 1 241 1 2 3 2 1 2 1 1 2 3 1 3 1 2 1 2 3 2 2 2 3 2 3 1 242 1 1 2 1 3 1 2 1 1 3 1 2 2 3 1 2 2 3 2 3 2 2 2 3 243 2 2 2 3 1 2 3 1 2 1 1 2 1 3 1 1 3 1 3 1 1 2 3 1 244 1 3 1 2 3 1 1 2 1 1 3 2 2 3 2 3 1 1 2 3 2 2 2 1 245 1 3 1 2 3 1 1 1 3 1 1 1 3 2 3 2 1 3 1 1 2 1 2 2 246 2 3 2 2 1 1 1 2 3 2 1 2 3 2 1 3 2 1 1 2 2 3 1 3 247 2 1 3 2 1 3 2 3 2 3 1 1 3 2 2 1 2 2 2 3 2 2 1 2 248 1 3 2 3 1 1 2 3 2 2 2 3 2 1 1 1 3 1 3 2 2 2 1 1 249 3 1 2 1 1 1 2 3 1 3 1 1 2 2 3 1 3 2 1 1 2 2 3 2 250 2 3 1 2 3 1 3 1 1 1 2 2 3 2 2 2 1 1 3 2 3 2 2 2 251 1 1 1 2 1 1 3 2 1 3 2 3 2 3 1 3 2 1 1 2 1 3 2 1 252 2 1 2 3 1 1 1 2 1 2 3 2 3 1 2 1 3 2 1 1 3 1 3 1 253 1 2 2 3 2 1 1 3 1 3 2 3 1 2 2 1 2 1 3 1 2 3 1 2 254 1 3 1 3 2 1 1 3 1 1 2 3 1 1 1 3 1 3 1 2 1 1 2 1 255 2 1 1 3 2 1 1 3 2 1 3 1 2 3 2 2 1 1 1 3 1 3 1 2 256 1 1 1 2 1 3 1 1 1 3 1 1 2 2 3 2 1 3 1 3 2 1 3 2 257 1 2 1 3 1 2 2 2 1 1 3 2 3 1 1 3 1 3 1 3 2 2 1 2 258 3 1 1 2 3 2 2 2 3 2 1 1 1 2 3 2 1 2 1 3 1 2 1 3 259 1 1 1 2 1 3 1 1 2 3 1 3 2 1 3 2 3 1 1 1 2 1 2 3 260 2 2 3 1 1 2 2 1 2 3 2 1 3 1 3 1 1 1 3 2 1 1 1 3 261 2 1 3 2 1 1 1 2 2 3 1 3 1 3 2 1 3 2 2 3 1 1 2 2 262 2 3 2 1 1 1 3 2 3 2 2 2 1 2 1 3 2 3 2 3 2 1 1 2 263 1 2 1 2 3 1 2 2 2 3 1 3 1 2 3 1 3 1 1 2 3 2 1 1 264 1 1 2 1 2 2 3 1 2 1 2 3 2 3 2 2 3 2 3 1 1 3 2 1 265 1 3 2 3 1 3 1 2 2 1 2 3 1 3 2 1 2 2 3 1 2 2 2 1 266 2 2 3 2 1 2 2 2 1 3 1 2 1 3 2 3 1 3 1 2 2 1 2 3 267 1 2 1 3 1 1 1 2 3 1 1 1 3 1 2 1 3 1 2 1 3 1 1 3 268 3 1 2 2 3 2 1 2 1 2 3 2 1 1 1 3 2 1 3 2 2 2 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2 1 2 3 1 2 3 2 2 3 2 2 2 3 2 1 2 2 1 2 1076 1 2 2 1 2 2 3 2 3 2 1 3 1 2 3 2 1 2 2 1 1 3 1 3 1077 3 2 2 1 3 1 1 1 3 1 2 2 2 1 3 1 1 3 2 2 1 3 2 2 1078 2 2 3 2 3 2 1 2 2 1 1 3 1 3 1 3 2 3 1 1 1 2 1 2 1079 3 2 2 2 1 1 3 1 2 1 3 1 1 1 3 1 3 2 3 1 2 2 2 1 1080 1 1 2 3 1 3 1 1 1 2 1 3 1 2 1 3 2 2 1 2 2 3 2 3 1081 2 3 1 1 2 2 3 1 1 2 1 1 3 1 1 2 2 2 3 2 2 3 2 3 1082 1 1 2 1 1 3 1 2 2 3 1 1 2 2 1 3 2 3 1 3 2 1 1 3 1083 1 1 2 3 2 2 2 3 1 3 1 3 1 2 2 2 1 3 2 1 1 1 3 1 1084 1 3 2 2 2 1 3 1 1 2 1 3 1 1 1 2 3 2 3 2 2 2 3 1 1085 2 1 2 1 1 3 2 1 1 3 2 3 2 2 1 1 3 1 2 2 2 3 1 3 1086 3 2 1 3 2 3 1 1 2 1 1 3 2 2 1 3 2 3 2 2 1 1 2 1 1087 1 1 3 2 3 2 3 2 2 1 1 1 3 2 1 1 1 2 3 2 1 3 1 2 1088 1 3 1 3 1 2 3 2 2 2 1 2 3 2 2 3 2 3 1 1 2 2 1 1 1089 1 3 2 2 3 1 1 2 1 2 2 3 1 2 3 1 2 1 1 3 1 1 3 1 1090 2 3 1 1 2 3 2 3 1 3 1 2 3 2 2 2 1 3 1 1 2 1 1 2 1091 1 1 2 1 1 2 3 1 2 3 2 1 1 3 2 2 2 3 1 3 2 2 2 3 1092 1 1 1 3 1 3 2 3 1 1 2 1 3 1 1 1 2 1 1 3 1 3 1 1 1093 1 1 2 1 1 1 3 2 2 1 2 2 3 1 3 1 3 1 3 2 2 2 1 3 1094 1 3 2 1 3 2 3 2 2 3 2 1 3 2 2 2 1 3 2 1 2 1 2 1 1095 3 2 1 1 3 1 1 2 3 2 1 2 2 1 3 1 2 1 2 2 2 3 2 3 1096 3 1 2 1 1 1 2 3 2 2 2 3 1 2 1 1 1 3 2 1 3 2 2 3 1097 1 2 1 3 2 1 2 3 2 1 2 3 2 3 2 3 1 1 3 1 2 2 2 1 1098 1 2 3 1 1 2 3 2 1 3 1 3 2 3 1 2 2 1 3 2 2 2 1 1 1099 3 2 1 3 2 1 2 2 2 1 3 2 3 1 2 3 2 1 1 3 1 1 2 1 1100 1 3 1 1 2 2 3 2 1 2 2 3 1 1 3 1 1 3 1 1 2 1 2 3 1101 2 2 2 1 2 1 3 1 1 2 2 3 1 3 1 3 1 1 3 2 2 1 1 3 1102 1 1 1 3 2 1 3 2 1 3 1 3 1 2 2 2 3 1 3 1 1 2 2 1 1103 2 2 2 1 1 1 3 1 1 1 3 2 1 2 2 3 2 1 1 3 1 3 2 3 1104 1 1 1 2 2 3 1 3 1 1 1 3 2 3 1 1 2 3 1 1 3 2 2 2 1105 1 1 3 1 1 1 2 1 1 3 2 1 2 3 1 2 1 3 2 1 3 2 1 3 1106 1 2 2 2 3 1 1 2 2 3 2 1 2 2 3 2 1 3 2 2 2 3 2 3 1107 1 1 3 1 3 1 1 2 1 1 2 3 2 1 3 1 3 1 2 1 2 1 1 3 1108 2 3 2 3 2 1 1 2 1 3 2 2 3 2 2 1 1 2 3 1 3 2 1 1 1109 2 1 2 1 3 2 2 3 2 1 3 2 2 2 1 3 1 2 3 1 1 2 3 2 1110 1 2 2 3 2 3 2 2 1 3 1 1 2 3 1 2 3 2 2 1 1 2 1 3 1111 3 2 2 2 3 2 1 2 1 3 2 1 2 2 2 3 1 2 2 3 1 2 3 2 1112 1 3 1 3 2 1 1 1 3 2 1 2 3 1 3 2 2 1 2 3 1 1 2 1 1113 3 1 1 1 3 2 2 2 1 1 3 2 3 1 2 3 2 1 2 1 2 2 3 2 1114 2 2 1 1 1 2 3 1 2 1 1 1 3 1 3 2 1 3 2 3 1 1 3 2 1115 2 2 1 1 1 2 3 2 3 2 3 1 3 1 1 3 1 2 3 1 1 2 T 1 1116 1 2 2 2 3 2 1 2 1 1 1 3 2 3 1 1 3 1 1 3 1 3 1 1 1117 2 3 1 2 2 1 3 2 1 2 2 2 3 2 3 1 1 3 1 3 1 2 2 2 1118 2 2 2 3 1 1 2 3 1 1 1 2 2 3 1 2 3 1 2 1 3 1 2 3 1119 1 3 1 3 2 1 1 3 1 2 2 1 1 3 1 1 2 1 1 3 1 1 1 3 1120 1 2 2 3 1 1 2 2 3 1 3 1 1 3 2 3 1 1 3 2 1 1 1 2 1121 2 2 2 1 3 1 3 1 1 3 2 1 2 2 3 2 2 2 3 1 1 1 3 1 1122 2 1 1 1 3 2 3 1 1 1 3 1 2 2 2 3 1 1 1 2 3 1 2 3 1123 3 1 1 1 3 2 2 1 3 1 3 1 1 1 2 3 2 1 3 1 1 1 2 2 1124 3 2 3 1 1 2 1 1 2 3 1 1 3 1 1 3 2 2 1 2 3 2 2 1 1125 2 2 3 2 3 1 1 2 1 1 1 3 2 1 3 1 2 3 2 3 2 2 1 2 1126 2 2 1 2 1 2 3 1 2 1 2 3 1 3 2 2 2 3 2 3 2 2 3 1 1127 2 2 3 1 2 2 2 3 2 3 2 3 1 3 2 1 2 2 1 3 2 2 1 2 1128 1 1 1 3 2 3 1 2 2 1 1 3 2 2 1 3 2 2 2 3 1 3 1 2 1129 2 2 3 2 1 2 2 2 3 2 1 2 1 1 2 3 2 2 3 1 1 3 1 3 1130 3 2 2 2 3 1 1 1 2 2 1 3 2 3 2 3 1 3 1 1 1 2 1 2 1131 1 1 2 3 2 2 3 1 3 1 2 2 3 1 2 1 1 2 3 2 2 3 1 1 1132 2 1 3 2 1 3 2 1 3 2 1 2 2 3 2 2 3 2 1 1 2 1 1 3 1133 3 2 2 3 2 1 1 2 2 2 3 1 3 2 3 2 2 1 3 2 2 1 2 2 1134 2 3 1 1 2 1 2 3 1 2 1 3 2 2 1 3 2 1 1 2 2 3 2 3 1135 2 3 1 2 1 3 2 1 2 3 2 2 2 3 2 3 1 2 2 1 1 1 3 1 1136 3 1 2 3 2 1 2 1 1 1 3 1 3 2 1 2 3 2 2 1 2 1 1 3 1137 1 3 2 3 1 3 1 2 2 2 1 3 1 1 3 1 2 3 2 2 1 2 2 1 1138 1 2 3 1 3 1 1 2 2 2 3 2 2 1 1 1 3 1 3 1 1 1 3 2 1139 1 1 1 3 1 1 2 2 1 3 2 1 2 3 1 2 1 3 1 2 3 1 3 1 1140 2 1 3 1 3 2 2 3 2 1 2 1 3 2 2 2 1 2 1 3 2 2 3 1 1141 3 2 1 3 1 1 2 3 1 2 2 3 2 2 2 1 3 1 1 3 1 2 2 2 1142 3 2 2 2 1 2 3 2 2 2 3 1 3 1 1 3 1 3 2 2 1 2 2 2 1143 2 1 3 1 1 3 2 2 2 3 1 1 1 3 2 2 1 2 2 3 1 2 2 3 1144 3 1 2 3 1 1 3 1 3 2 1 2 2 2 3 2 2 1 2 1 2 3 2 1 1145 3 1 2 3 1 1 2 1 2 1 3 2 1 1 3 2 1 2 2 3 1 3 2 1 1146 2 1 3 2 3 1 2 3 1 1 1 2 2 2 3 1 3 1 2 1 3 1 2 1 1147 3 1 1 1 3 1 1 1 2 2 3 1 1 3 1 3 2 2 2 3 1 2 1 2 1148 1 2 2 2 3 1 3 2 1 2 2 2 3 2 3 2 1 2 2 3 1 1 2 3 1149 1 2 3 1 3 2 2 3 1 1 1 2 2 2 3 1 1 3 2 1 2 2 3 2 1150 2 2 1 1 2 1 3 2 3 1 3 1 3 1 3 2 1 2 1 2 3 2 1 1 1151 1 2 2 1 1 3 1 3 1 3 2 3 1 3 2 1 1 1 2 3 2 1 1 1 1152 1 1 3 1 1 2 1 3 1 2 3 1 3 1 2 2 1 3 1 1 1 2 1 3 1153 1 3 2 2 2 1 1 1 3 1 3 2 2 1 3 1 1 2 2 3 1 1 1 3 1154 3 2 1 1 3 1 2 2 2 3 2 2 3 1 1 2 1 1 1 3 1 1 3 1 1155 1 3 1 3 1 1 1 3 1 1 3 2 2 1 1 1 3 2 3 1 2 1 2 2 1156 2 1 1 2 1 3 1 3 1 1 3 1 3 1 2 3 2 1 2 3 1 1 2 1 1157 2 2 1 2 2 1 3 2 3 1 2 1 1 3 2 3 1 1 3 2 2 2 1 3 1158 1 2 1 1 2 3 2 1 1 1 3 1 2 3 1 3 2 2 2 1 2 3 1 3 1159 2 2 3 1 2 2 2 3 1 3 1 3 2 2 3 1 2 1 1 3 1 2 2 2 1160 1 2 3 1 2 2 1 2 2 3 2 3 2 3 2 1 3 1 1 2 2 1 3 1 1161 2 1 2 1 1 1 3 1 2 1 2 1 3 2 1 3 1 2 3 1 2 3 2 3 1162 2 2 2 1 3 2 2 3 1 3 1 2 3 1 1 3 2 2 1 2 2 1 3 1 1163 1 2 2 3 1 1 2 2 3 1 2 1 2 1 3 2 3 2 1 1 1 3 2 3 1164 3 1 1 3 1 1 1 3 1 2 2 1 2 2 3 2 1 2 2 3 1 3 2 2 1165 1 2 2 3 1 3 2 3 2 1 3 2 3 1 2 2 2 1 3 1 1 1 2 1 1166 1 1 2 1 1 1 3 2 3 2 2 2 1 1 3 1 3 2 1 3 1 3 2 1 1167 3 2 1 3 1 3 1 2 1 1 2 2 3 1 2 3 2 3 2 1 1 2 2 2 1168 In Table IIA, each of the numerals 1 to 3 (numeric identifiers) represents a nucleotide base and the pattern of numerals 1 to 3 of the sequences the above list corresponds to the pattern of nucleotide bases present in the oligonucleotides of Table II, which oligonucleotides have been found to be non-cross-hybridizing, as described further in the detailed examples. Each nucleotide base is selected from the group of nucleotide bases consisting of A, C, G, and T/U. A particularly preferred embodiment of the invention, in which a specific base is assigned to each numeric identifier is shown in Table II, below.

In one broad aspect, the invention is a composition comprising molecules for use as tags or tag complements wherein each molecule comprises an oligonucleotide selected from a set of oligonucleotides based on a group of sequences as specified by numeric identifiers set out in Table IIA. In the sequences, each of 1 to 3 is a nucleotide base selected to be different from the others of 1 to 3 with the proviso that up to three nucleotide bases of each sequence can be substituted with any nucleotide base provided that:

-   -   for any pair of sequences of the set:     -   M1≦16, M2≦13, M3≦20, M4≦16, and M5≦19, where:     -   M1 is the maximum number of matches for any alignment in which         there are no internal indels;     -   M2 is the maximum length of a block of matches for any         alignment;     -   M3 is the maximum number of matches for any alignment having a         maximum score;     -   M4 is the maximum sum of the lengths of the longest two blocks         of matches for     -   any alignment of maximum score; and     -   M5 is the maximum sum of the lengths of all the blocks of         matches having a length of at least 3, for any alignment of         maximum score; wherein:         -   the score of an alignment is determined according to the             equation (A×m)−(B×mm)−(C×(og+eg))−(D−eg)), wherein:             -   for each of (i) to (iv):                 -   (i) m=6, mm=6, og=0 and eg=6,                 -   (ii) m=6, mm=6, og=5 and eg=1,                 -   (iii) m=6, mm=2, og=5 and eg=1, and                 -   (iv) m=6, mm=6., og=6 and eg=0,             -   A is the total number of matched pairs of bases in the                 alignment;             -   B is the total number of internal mismatched pairs in                 the alignment;             -   C is the total number of internal gaps in the alignment;                 and.             -   D is the total number of internal indels in the                 alignment minus             -   the total number of internal gaps in the alignment; and         -   wherein the maximum score is determined separately for each             of (i), (ii), (iii) and (iv).

An explanation of the meaning of the parameters set out above is given in the section describing detailed embodiments.

In another broad aspect the invention is a composition containing molecules for use as tags or tag complements wherein each molecule comprises an oligonucleotide selected from a set of oligonucleotides based on a group of sequences as set out in Table IIA wherein each of 1 to 3 is a nucleotide base selected to be different from the others of 1 to 3 with the proviso that up to three nucleotide bases of each sequence can be substituted with any nucleotide base provided that:

-   for any pair of sequences of the set:     -   M1≦19, M2≦17, M3≦21, M4≦18, and M5≦20, where:         -   M1 is the maximum number of matches for any alignment in             which there are no internal indels;         -   M2 is the maximum length of a block of matches for any             alignment;         -   M3 is the maximum number of matches for any alignment having             a maximum score;         -   M4 is the maximum sum of the lengths of the longest two             blocks of matches for any alignment of maximum score; and         -   M5 is the maximum sum of the lengths of all the blocks of             matches having a length of at least 3, for any alignment of             maximum score; wherein             -   the score of an alignment is determined according to the                 equation (A×m)−(B×mm)−(C×(og+eg))−(D×eg)), wherein:                 -   for each of (i) to (iv)                 -    (i) m=6, mm=6, og=0 and eg=6,                 -    (ii) m=6, mm=6, og=5 and eg=1,                 -    (iii) m=6, mm=2, og=S and eg=1, and                 -    (iv) m=6, mm=6, og=6 and eg=0,                 -   A is the total number of matched pairs of bases in                     the alignment;                 -   B is the total number of internal mismatched pairs                     in the alignment;                 -   C is the total number of internal gaps in the                     alignment; and                 -   D is the total number of internal indels in the                     alignment minus the total number of internal gaps in                     the alignment; and             -   wherein the maximum score is determined separately for                 each of (i), (ii), (iii) and (iv).

In another broad aspect, the invention is a composition comprising molecules for use as tags or tag complements wherein each molecule comprises an oligonucleotide selected from a set of oligonucleotides based on a group of sequences set out in Table IIA wherein each of 1 to 3 is a nucleotide base selected to be different from the others of 1 to 3 with the proviso that up to three nucleotide bases of each sequence can be substituted with any nucleotide base provided that:

-   for any pair of sequences of the set:     -   M1≦19, M2≦17, M3≦21, M4≦18, and M5≦20, where:         -   M1 is the maximum number of matches for any alignment in             which there are n internal indels;         -   M2 is the maximum length of a block of matches for any             alignment;         -   M3 is the maximum number of matches for any alignment having             a maximum score;         -   M4 is the maximum sum of the lengths of the longest two             blocks of matches for any alignment of maximum score; and         -   M5 is the maximum sum of the lengths of all the blocks of             matches having a length of at least 3, for any alignment of             maximum score, wherein:             -   the score of an alignment is determined according to the                 equation 3A−B−3C−D, wherein:                 -   A is the total number of matched pairs of bases in                     the alignment;                 -   B is the total number of internal mismatched pairs                     in the alignment;                 -   C is the total number of internal gaps in the                     alignment; and                 -   D is the total number of internal indels in the                     alignment minus                 -   the total number of internal gaps in the alignment;                     and

In preferred aspects, the invention provides a composition in which, for the group of 24mer sequences in which 1=A, 2=T and 3=G, under a defined set of conditions in which the maximum degree of hybridization between a sequence and any complement of a different sequence of the group of. 24mer sequences does not exceed 30% of the degree of hybridization between said sequence and its complement, for all said oligonucleotides of the composition, the maximum degree of hybridization between an oligonucleotide and a complement of any other oligonucleotide of the composition does not exceed 50% of the degree of hybridization of the oligonucleotide and its complement.

More preferably, the maximum degree of hybridization between a sequence and any complement of a different sequence does not exceed 30% of the degree of hybridization between said sequence and its complement, the degree of hybridization between each sequence and its complement varies by a factor of between 1 and up to 10, more preferably between 1 and up to 9, more preferably between 1 and up to 8, more preferably between 1 and up to 7, more preferably between 1 and up to 6, and more preferably between 1 and up to 5.

It is also preferred that the maximum degree of hybridization between a sequence and any complement of a different sequence does not exceed 25%, more preferably does not exceed 20.%, more preferably does not exceed 15%, more preferably does not exceed 10%, more preferably does not exceed 5%.

Even more preferably, the above-referenced defined set of conditions results in a level of hybridization that is the same as the level of hybridization obtained when hybridization conditions include 0.2 M NaCl, 0.1 M Tris, 0.08% Triton X-100, pH 8.0 at 37° C.

In the composition, the defined set of conditions can include the group of 24mer sequences being covalently linked to beads.

In a particular preferred aspect, for the group of 24mers the maximum degree of hybridization between a sequence and any complement of a different sequence does not exceed 15% of the degree of hybridization between said sequence and its complement and the degree of hybridization between each sequence and its complement varies by a factor of between 1 and up to 9, and for all oligonucleotides of the set, the maximum degree of hybridization between an oligonucleotide and a complement of any other oligonucleotide of the set does not exceed 20% of the degree of hybridization of the oligonucleotide and its complement.

It is possible that each 1 is one of A, T/U, G and C; each 2 is one of A, T/U, G and C; and each 3 is one of A, T/U, G and C; and each of 1, 2 and 3 is selected so as to be different from all of the others of 1, 2 and 3. More preferably, 1 is A or T/U, 2 is A or T/U and 3 is G or C. Even more preferably, 1 is A, 2 is T/U, and 3 is G.

In certain preferred composition, each of the oligonucleotides is from twenty-two to twenty-six bases in length, or from twenty-three to twenty-five, and preferably, each oligonucleotide is of the same length as every other said oligonucleotide.

In a particularly preferred embodiment, each oligonucleotide is twenty-four bases in length.

It is preferred that no oligonucleotide contains more than four contiguous bases that are identical to each other.

It is also preferred that the number of G's in each oligonucleotide does not exceed L/4 where L is the number of bases in said sequence.

For reasons described below, the number of G's in each said oligonucleotide is preferred not to vary from the average number of G's in all of the oligonucleotides by more than one. Even more preferably, the number of G's in each said oligonucleotide is the same as-every other said oligonucleotide. In the embodiment disclosed below in which oligonucleotides were tested, the sequence of each was twenty-four bases in length and each oligonucleotide contained 6 G's.

It is also preferred that, for each nucleotide, there is at most six bases other than G between every pair of neighboring pairs of G's.

Also, it is preferred that, at the 5′-end of each oligonucleotide at least one of the first, second, third, fourth, fifth, sixth and seventh bases of the sequence of the oligonculeotide is a G. Similarly, it is preferred, at the 3′-end of each oligonucleotide that at least one of the first, second, third, fourth, fifth, sixth and seventh bases of the sequence of the oligonucleotide is a G.

It is possible to have sequence compositions that include one hundred and sixty said molecules, or that include one hundred and seventy said molecules, or that include one hundred and eighty said molecules, or that include one hundred and ninety said molecules, or that include two hundred said molecules, or that include two hundred and twenty said molecules, or that include two hundred and forty said molecules, or that include two hundred and sixty said molecules, or that include two hundred and eighty said molecules, or that include three hundred said molecules, or that include four hundred said molecules, or that include five hundred said molecules, or that include six hundred said molecules, or that include seven hundred said molecules, or that include eight hundred said molecules, or that include nine hundred said molecules, or that include one thousand said molecules.

It is possible, in certain applications, for each molecule to be linked to a solid phase support so as to be distinguishable from a mixture containing other of the molecules by hybridization to its complement. Such a molecule can be linked to a defined location on a solid phase support such that the defined location for each molecule is different than the defined location for different others of the molecules.

In certain embodiments, each solid phase support is a microparticle and each said molecule is covalently linked to a different microparticle than each other different said molecule.

In another broad aspect, the invention is a composition comprising a set of 150 molecules for use as tags or tag complements wherein each molecule includes an oligonucleotide having a sequence of at least sixteen nucleotide bases wherein for any pair of sequences of the set:

-   -   M1≦19/24×L1, M2≦17/24×L1, M3≦21/24×L1, M4≦18/24×L1,     -   M5≦20/24×L1, where L1 is the length of the shortest sequence of         the pair, where:         -   M1 is the maximum number of matches for any alignment of the             pair of sequences in which there are no internal indels;         -   M2 is the maximum length of a block of matches for any             alignment of the pair of sequences;         -   M3 is the maximum number of matches for any alignment of the             pair of sequences having a maximum score;         -   M4 is the maximum sum of the lengths of the longest two             blocks of matches for any alignment of the pair of sequences             of maximum score; and         -   M5 is the maximum sum of the lengths of all the blocks of             matches having length of at least 3, for any alignment of             the pair of sequences of maximum score, wherein:             -   the score of an alignment is determined according to the                 equation (A×m)−(B×mm)−(C×(og+eg))−(D×eg)), wherein:                 -   for each of (i) to (iv):                 -    (i) m=6, mm=6, og=0 and eg=6,                 -    (ii) m=6, mm=6, og=5 and eg=1,                 -    (iii) m=6, mm=2, og=5 and eg=1, and                 -    (iv) m=6, mm=6, og=6 and eg=0,                 -   A is the total number of matched pairs of bases in                     the alignment;                 -   B is the total number of internal mismatched pairs                     in the alignment;                 -   C is the total number of internal gaps in the                     alignment; and                 -   D is the total number of internal indels in the                     alignment minus the total number of internal gaps in                     the alignment; and             -   wherein the maximum score is determined separately for                 each of (i), (ii), (iii) and (iv).

In yet another broad aspect, the invention is a composition that includes a set of 150 molecules for use as tags or tag complements wherein each molecule has an oligonucleotide having a sequence of at least sixteen nucleotide bases wherein for any pair of sequences of the set:

-   -   M1≦19, M2≦17, M3≦21, M4≦18, and M5≦20, where:         -   M1 is the maximum number of matches for any alignment of the             pair of sequences in which there are no internal indels;         -   M2 is the maximum length of a block of matches for any             alignment of the pair of sequences;         -   M3 is the maximum number of matches for any alignment of the             pair of sequences having a maximum score;         -   M4 is the maximum sum of the lengths of the longest two             blocks of matches for any alignment of the pair of sequences             of maximum score; and         -   M5 is the maximum sum of the lengths of all the blocks of             matches having a length of at least 3, for any alignment of             the pair of sequences of maximum score, wherein:             -   the score of a said alignment is determined according to                 the equation 3A−B−3C−D, wherein:                 -   A is the total number of matched pairs of bases in                     the alignment;                 -   B is the total number of internal mismatched pairs                     in the alignment;                 -   C is the total number of internal gaps in the                     alignment; and                 -   D is the total number of internal indels in the                     alignment minus the total number of internal gaps in                     the alignment.

In certain embodiments of the invention., each sequence of a composition has up to fifty bases. More preferably, however, each sequence is between sixteen and forty bases in length, or between sixteen and thirty-five bases in length, or between eighteen and thirty bases in length, or between twenty and twenty-eight bases in length, or between twenty-one and twenty-seven bases in length, or between twenty-two and twenty-six bases in length.

Often, each sequence is of the same length as every other said sequence. In particular embodiments disclosed-herein, each sequence is twenty-four bases in length.

Again, it can be preferred that no sequence contains more than four contiguous bases that are identical to each other, etc., as described above.

In certain preferred embodiments, the composition is such that, under a defined set of conditions, the maximum degree of hybridization between an oligonucleotide and any complement of a different oligonucleotide of the composition does not exceed about 30% of the degree of hybridization between said oligonucleotide and its complement, more preferably 20%, more preferably 15%, more preferably 10%, more preferably 6%.

Preferably, the set of conditions results in a level of hybridization that is the same as the level of hybridization obtained when hybridization conditions include 0.2 M NaCl, 0.1 M Tris, 0.08% Triton X-100, pH 8.0 at 37° C., and the oligonucleotides are covalently linked to microparticles. Of course it is possible that these specific conditions be used for determining the level of hybridization.

It is also preferred that under such a defined set of conditions, the degree of hybridization between each oligonucleotide and its complement varies by a factor of between 1 and up to 8, more preferably up to 7, more preferably up to 6, more preferably up to 5. In a particular disclosed embodiment, the observed variance in the degree of hybridization was a factor of only 5.3, i.e., the degree of hybridization between each oligonucleotide and its complement varied by a factor of between 1 and 5.6.

In certain preferred embodiments, under the defined set of conditions, the maximum degree of hybridization between a said oligonucleotide and any complement of a different oligonucleotide of the composition does not exceed about 15%, more preferably 10%, more preferably 6%.

In one preferred embodiment, the set of conditions results in a level of hybridization that is the same as the level of hybridization obtained when hybridization conditions include 0.2 M NaCl, 0.1 M Tris, 0.08% Triton X-100, pH 8.0 at 37° C., and the oligonucleotides are covalently linked to microparticles.

Also, under the defined set of conditions, it is preferred that the degree of hybridization between each oligonucleotide and its complement varies by a factor of between 1 and up to 8, more preferably up to 7, more preferably up to 6, more preferably up to 5.

Any composition of the invention can include one hundred and sixty of the oligonucleotide molecules, or one hundred and seventy of the oligonucleotide molecules, or one hundred and eighty of the oligonucleotide molecules, or one hundred and ninety of the oligonucleotide molecules, or two hundred of the oligonucleotide molecules, or two hundred and twenty of the oligonucleotide molecules, or two hundred and forty of the oligonucleotide molecules, or two hundred and sixty of the oligonucleotide molecules, or two hundred and eighty of the oligonucleotide molecules, or three hundred of the oligonucleotide molecules, or four hundred of the oligonucleotide molecules, or five hundred of the oligonucleotide molecules, or six hundred of the oligonucleotide molecules, or seven hundred of the oligonucleotide molecules, or eight hundred of the oligonucleotide molecules, or nine hundred of the oligonucleotide molecules, or one thousand or more of the oligonucleotide molecules.

A composition of the invention can be a family of tags, or it can be a family of tag complements.

An oligonucleotide molecule belonging to a family of molecules of the invention can have incorporated thereinto one more analogues of nucleotide bases, preference being given those that undergo normal Watson-Crick base pairing.

The invention includes kits for sorting and identifying polynucleotides. Such a kit can include one or more solid phase supports each having one or more spatially discrete regions, each such region having a uniform population of substantially identical tag complements covalently attached. The tag complements are made up of a set of oligonucleotides of the invention.

The one or more solid phase supports can be a planar substrate in which the one or more spatially discrete regions is a plurality of spatially addressable regions.

The tag complements can also be coupled to microparticles. Microparticles preferably each have a diameter in the range of from 5 to 40 μm.

Such a kit preferably includes microparticles that are spectrophotometrically unique, and therefore distinguisable from each other according to conventional laboratory techniques. Of course for such kits to work, each type of microparticle would generally have only one tag complement associated with it, and usually there would be a different oligonucleotide tag complement associated with (attached to) each type of microparticle.

The invention includes methods of using families of oligonucleotides of the invention.

One such method is of analyzing a biological sample containing a biological sequence for the presence of a mutation or polymorphism at a locus of the nucleic acid. The method includes:

-   (A) amplifying the nucleic acid molecule in the presence of a first     primer having a 5′-sequence having the sequence of a tag     complementary to the sequence of a tag complement belonging to a     family of tag complements of the invention to form an amplified     molecule with a 5′-end with a sequence complementary to the sequence     of the tag; -   (B) extending the amplified molecule in the presence of a polymerase     and a second primer having 5′-end complementary the 3′-end of the     amplified sequence, with the 3′-end of the second primer extending     to immediately adjacent said locus, in the presence of a plurality     of nucleoside triphosphate derivatives each of which is: (i) capable     of incorporation during transciption by the polymerase onto the     3′-end of a growing nucleotide strand; (ii) causes termination of     polymerization; and (iii) capable of differential detection, one     from the other, wherein there is a said derivative complementary to     each possible nucleotide present at said locus of the amplified     sequence; -   (C) specifically hybridizing the second primer to a tag complement     having the tag complement sequence of (A); and -   (D) detecting the nucleotide derivative incorporated into the second     primer in (B) so as to identify the base located at the locus of the     nucleic acid.

In another method of the invention, a biological sample containing a plurality of nucleic acid molecules is analyzed for the presence of a mutation or polymorphism at a locus of each nucleic acid molecule, for each nucleic acid molecule. This method includes steps of:

-   (A) amplifying the nucleic acid molecule in the presence of a first     primer having a 5′-sequence having the sequence of a tag     complementary to the sequence of a tag complement belonging to a     family of tag complements of the invention to form an amplified     molecule with a 5′-end with a sequence complementary to the sequence     of the tag; -   (B) extending the amplified molecule in the presence of a polymerase     and a second primer having 5′-end complementary the 3′-end of the     amplified sequence, the 3′-end of the second primer extending to     immediately adjacent said locus, in the presence of a plurality of     nucleoside triphosphate derivatives each of which is: (i) capable of     incorporation during transciption by the polymerase onto the 3′-end     of a growing nucleotide strand; (ii) causes termination of     polymerization; and (iii) capable of differential detection, one     from the other, wherein there is a said derivative complementary to     each possible nucleotide present at said locus of the amplified     molecule; -   (C) specifically hybridizing the second primer to a tag complement     having the tag complement sequence of (A); and -   (D) detecting the nucleotide derivative incorporated into the second     primer in (B) so as to identify the base located at the locus of the     nucleic acid;     wherein each tag of (A) is unique for each nucleic acid molecule and     steps (A) a: -   (B) are carried out with said nucleic molecules in the presence of     each other.

Another method includes analyzing a biological sample that contains a plurality of double stranded complementary nucleic acid molecules for the presence of a mutation or polymorphism at a locus of each nucleic acid molecule, for each nucleic acid molecule. The method includes steps of:

-   (A) amplifying the double stranded molecule in the presence of a     pair of first primers, each primer having an identical 5′-sequence     having the sequence of a tag complementary to the sequence of a tag     complement belonging to a family of tag complements of the invention     to form amplified molecules with 5′-ends with a sequence     complementary to the sequence of the tag; -   (B) extending the amplified molecules in the presence of a     polymerase and a p of second primers each second primer having a     5′-end complementary a 3′-end of the amplified sequence, the 3′-end     of each said second primer extending to immediately adjacent said     locus, in the presence of a plurality of nucleoside triphosphate     derivatives each of which is: (i) capable of incorporation during     transciption by the polymerase onto the 3′-end of a growing     nucleotide strand; (ii) causes termination of polymerization;     and (iii) capable of differential detection, one from the other; -   (C) specifically hybridizing each of the second primers to a tag     complement having the tag complement sequence of (A); and -   (D) detecting the nucleotide derivative incorporated into the second     primers in (B) so as to identify the base located at said locus;     wherein the sequence of each tag of (A) is unique for each nucleic     acid molecule and steps (A) and (B) are carried out with said     nucleic molecules in the presence of each other.

In yet another aspect, the invention is a method of analyzing a biological sample containing a plurality of nucleic acid molecules for the presence of a mutation or polymorphism at a locus of each nucleic acid molecule, for each nucleic acid molecule, the method including steps of:

-   (a) hybridizing the molecule and a primer, the primer having a     5′-sequence having the sequence of a tag complementary to the     sequence of a tag complement belonging to a family of tag     complements of the invention and a 3′-end extending to immediately     adjacent the locus; -   (b) enzymatically extending the 3′-end of the primer in the presence     of a plurality of nucleoside triphosphate derivatives each of which     is: (i) capable of enzymatic incorporation onto the 3′-end of a     growing nucleotide strand; (ii) causes termination of said     extension; and (iii) capable of differential detection, one from the     other, wherein there is a said derivative complementary to each     possible nucleotide present at said locus; -   (c) specifically hybridizing the extended primer formed in step (b)     to a tag complement having the tag complement sequence of (a); and -   (d) detecting the nucleotide derivative incorporated into the primer     in step (b) so as to identify the base located at the locus of the     nucleic a molecule;     wherein each tag of (a) is unique for each nucleic acid molecule and     steps (a) a (b) are carried out with said nucleic molecules in the     presence of each other.

The derivative can be a dideoxy nucleoside triphosphate.

Each respective complement can be attached as a uniform population of substantially identical complements in spacially discrete regions on one or more solid phase support(s).

Each tag complement can include a label, each such label being different for respective complements, and step (d) can include detecting the presence of the different labels for respective hybridization complexes of bound tags and tag complements.

Another aspect of the invention includes a method of determining the presence of a target suspected of being contained in a mixture. The method includes the steps of:

-   (i) labelling the target with a first label; -   (ii) providing a first detection moiety capable of specific binding     to the target and including a first tag; -   (iii) exposing a sample of the mixture to the detection moiety under     conditions suitable to permit (or cause) said specific binding of     the molecule and target; -   (iv) providing a family of suitable tag complements of the invention     wherein the family contains a first tag complement having a sequence     complementary to that of the first tag; -   (v) exposing the sample to the family of tag complements     under-conditions suitable to permit (or cause) specific     hybridization of the first tag and its tag complement; -   (vi) determining whether a said first detection moiety hybridized to     a first s tag complement is bound to a said labelled target in order     to determine t presence or absence of said target in the mixture.

Preferably, the first tag complement is linked to a solid support at a specific location of the support and step (vi) includes detecting the presence of the first label at said specified location.

Also, the first tag complement can include a second label and step (vi) includes detecting the presence of the first and second labels in a hybridized complex of the moiety and the first tag complement.

Further, the target can be selected from the group consisting of organic molecules, antigens, proteins, polypeptides, antibodies and nucleic acids. The target can be an antigen and the first molecule can be an antibody specific for that antigen.

The antigen is usually a polypeptide or protein and the labelling step can include conjugation of fluorescent molecules, digoxigenin, biotinylation and the like.

The target can be a nucleic acid and the labelling step can include incorporation of fluorescent molecules, radiolabelled nucleotide, digoxigenin, biotinylation and the like.

Another aspect of the invention includes detecting the presence of a target nucleic acid molecule using the Invader Assay, which is described in detail in U.S. Pat. No. 5,985,557 issued Nov. 16, 1999, incorporated herein by reference. The sequences of the present invention are incorporated into the 3′ portion of one of the two oligonucleotide probes that will eventually be cleaved by a Cleavase enzyme and captured by its complement which may be attached on a solid phase support in a microarray format.

Another aspect of the invention includes a method of analyzing a biological sample comprising a plurality of target nucleic acid molecules for the presence of a mutation or polymorphism at a locus of each target nucleic acid molecule using the Invader Assay. Again, the sequences of the present invention are incorporated into the 3′ portion of one of the two probes that will eventually be cleaved by a Cleavase enzyme and detected by using the cleaved sequence's complement, which may be attached on a solid phase support such as in a microarray format.

Another aspect of the invention incorporates the use of a second target nucleic acid sequence, wherein the second target nucleic acid sequence comprises a synthetic nucleic acid. The synthetic nucleic acid may further comprise at least one hairpin loop. The construction and use of such nucleic acid sequences with hairpin loops has been described in detail in U.S. Pat. No. 5,770,365 issued Jun. 23, 1998 and International Publication WO 01/94625A2 published Dec. 13, 2001.

The present invention capitalizes on the exquisite specificity of the Invader Assay and the minimally cross-hybridizing sequences of the present invention such that simultaneous use of multiple hybridization probes in a single experiment is now possible. The methods and compositions of the present invention allow for accurate and homogenous genotyping of a plurality of distinct nucleic acid in a single experiment. The methods and compositions of the present invention are flexible enough to extend to novel loci with little optimization the features of both the Invader Assay and the sequences of the present invention lend the technology to automation.

DETAILED DESCRIPTION OF THE INVENTION

Figures

Reference is made to the attached figures in which,

FIGS. 1A and 1B illustrate results obtained in the cross-hybridization experiments described in Example 1. FIG. 1A shows the hybridization pattern found when a microarray containing all 100 probes (SEQ ID NOs:1 to 100 of Table I) was hybridized with a 24mer oligonucleotide having the complementary sequence to SEQ ID NO:3 of Table I(target). FIG. 1B shows the pattern observed when a similar array was hybridized with a mix of all 100 targets, i.e., oligonucleotides having the sequences complementary to SEQ ID NOs:1 to 100 of Table 1.

FIG. 2 shows the intensity of the signal (MFI) for each perfectly matched sequence (indicated in Table I) and its complement obtained as described in Example 3.

FIG. 3 is a three dimensional representation showing cross-hybridization observed for the sequences of FIG. 2 as described in Example 3. The results shown in FIG. 2 are reproduced along the diagonal of the drawing.

FIG. 4 is illustrative of results obtained for an individual target (SEQ ID NO:23 of Table I, target No. 16) when exposed to the 100 probes of Example 3. The MFI for each bead is plotted.

FIG. 5 illustrates generally the steps followed to obtain a family of sequences of the present invention;

FIG. 6 shows the intensity of the signal (MFI) for each perfectly matched sequence (probe sequence indicated in Table II) and its complement (target at 50 fmol) obtained as described in Example 4;

FIG. 7 is a three dimensional representation showing cross-hybridization observed for the sequences of FIG. 6 as described in Example 4. The results shown in FIG. 6 are reproduced along the diagonal of the drawing;

FIG. 8 is illustrative of the results obtained for an individual target (Table II, SEQ ID No: 90, target No. 90) when exposed to the 100 probes of Example 4. The MFI for each bead is plotted.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a method for sorting complex mixtures of molecules by the use of families of oligonucleotide sequence tags. The families of oligonucleotide sequence tags are designed so as to provide minimal cross hybridization during the sorting process. Thus any sequence within a family of sequences will not cross hybridize with any other sequence derived from that family under appropriate hybridization conditions known by those skilled in the art. The invention is particularly useful in highly parallel processing of analytes.

Families of Oligonucleotide Sequence Tags

The present invention includes a family of 24mer polynucleotides, that have been demonstrated to be minimally cross-hybridizing with each other. This family of polynucleotides is thus useful as a family of tags, and their complements as tag complements.

The oligonucleotide sequences that belong to families of sequences that do not exhibit cross hybridization behavior can be derived by computer programs (described in U.S. Provisional Patent Application No. 60/181,563 filed Feb. 10, 2000). The programs use a method of generating a maximum number of minimally cross-hybridizing polynucleotide sequences that can be summarized as follows. First, a set of sequences of a given length are created based on a given number of block elements. Thus, if a family of polynucleotide sequences 24 nucleotides (24mer) in length is desired from a set of 6 block elements, each element comprising 4 nucleotides, then a family of 24mers is generated considering all positions of the 6 block elements. In this case, there will be 6⁶ (46,656) ways of assembling the 6 block elements to generate all possible polynucleotide sequences 24 nucleotides in length.

Constraints are imposed on the sequences and are expressed as a set of rules on the identities of the blocks such that homology between any two sequences will not exceed the degree of homology desired between these two sequences. All polynucleotide sequences generated which obey the rules are saved. Sequence comparisons are performed in order to generate an incidence matrix. The incidence matrix is presented as a simple graph and the sequences with the desired property of being minimally cross hybridizing are found from a clique of the simple graph, which may have multiple cliques. Once a clique containing a suitably large number of sequences is found, the sequences are experimentally tested to determine if it is a set of minimally cross hybridizing sequences. This method has been used to obtain the 100 non cross-hybridizing tags of Table I that are the subject of Example 1.

The method includes a rational approach to the selection of groups of sequences that are used to describe the blocks. For example there are n⁴ different tetramers that can be obtained from n different nucleotides, non-standard bases or analogues thereof. In a more preferred embodiment there are 44 or 256 possible tetramers when natural nucleotides are used. More preferably 81 possible tetramers when only 3 bases are used A, T and G. Most preferably 32 different tetramers when all sequences have only one G.

Block sequences can be composed of a subset of natural bases most preferably A, T and G. Sequences derived from blocks that are deficient in one base possess useful characteristics, for example, in reducing potential secondary structure formation or reduced potential for cross hybridization with nucleic acids in nature. Sets of block sequences that are most preferable in constructing families of non cross hybridizing tag sequences should contribute approximately equivalent stability to the formation of the correct duplex as all other block sequences of the set. This should provide tag sequences that behave isothermally. This can be achieved, for example, by maintaining a constant base composition for all block sequences such as one G and three A's or T's for each block sequence. Preferably, non-cross hybridizing sets of block sequences will be comprised from blocks of sequences that are isothermal. The block sequences should be different from each other by at least one mismatch. Guidance for selecting such sequences is provided by methods for selecting primer and or probe sequences that can be found in published techniques (Robertson et al., Methods Mol Biol; 98:121-54 (1998); Rychlik et al, Nucleic Acids Research, 17:8543-8551 (1989); Breslauer et al., Proc Natl Acad. Sci., 83:3746-3750 (1986)) and the like. Additional sets of sequences can be designed by extrapolating on the original family of non cross hybridizing sequences by simple methods known to those skilled in the art.

A preferred family of 100 tags is shown as SEQ ID NOs:1 to 100 in Table I. Characterization of the family of 100 sequence tags was performed to determine the ability of these sequences to form specific duplex structures with their complementary sequences and to assess the potential for cross hybridization. The 100 sequences were synthesized and spotted onto glass slides where they were coupled to the surface by amine linkage. Complementary tag sequences were Cy3-labeled and hybridized individually to the array containing the family of 100 sequence tags. Formation of duplex structures was detected and quantified for each of the positions on the array. Each of the tag sequences performed as expected, that is the perfect match duplex was formed in the absence of significant cross hybridization under stringent hybridization conditions. The results of a sample hybridization are shown in FIG. 1. FIG. 1 a shows the hybridization pattern seen when a microarray containing all 100 probes was hybridized with the target complementary to probe 181234. The 4 sets of paired spots correspond to the probe complementary to the target. FIG. 1 b shows the pattern seen when a similar array was hybridized with a mix of all 100 targets. These results indicate that the family of sequences which is the subject of this patent can be used as a family of non-cross hybridizing (tag) sequences.

The family of 100 non-cross-hybridizing sequences can be expanded by incorporating additional tetramer sequences that are used in constructing further 24mer oligonucleotides. In one example, four additional words were included in the generation of new sequences to be considered for inclusion as non-cross talkers in a family of sequences that were obtained from the above method using 10 tetramers. In this case, the four additional words were selected to avoid potential homologies with all potential combinations of other words: YYXW (TTAG); WYYX (GTTA); XYXW (ATAG) and WYYY (GTTT). The total number of sequences containing six words using the 14 possible words is 14⁶ or 7,529,536. These sequences were screened to eliminate sequences that contain repetitive regions that present potential hybridization problems such as four or more of a similar base (e.g., AAAA or TTTT) or pairs of G's. Each of these sequences was compared to the sequence set of the original family of 100 non-cross-hybridizing sequences (SEQ ID NOs:1 to 100). Any new sequence that contained a minimal threshold of homology (that does not include the use of insertions or deletions) such as 15 or more matches with any of the original family of sequences was eliminated. In other words, if it was possible to align a new sequence with one or more of the original 100 sequences so as to obtain a maximum simple homology of 15/24 or more, the new sequence was dropped. “Simple homology” between a pair of sequences is defined here as the number of pairs of nucleotides that are matching (are the same as each other) in a comparison of two aligned sequences divided by the total number of potential matches. “Maximum simple homology” is obtained when two sequences are aligned with each other so as to have the maximum number of paired matching nucleotides. In any event, the set of new sequences so obtained was referred to as the “candidate sequences”. One of the candidate sequences was arbitrarily chosen and referred togas sequence 101. All the candidate sequences were checked against sequence 101, and sequences that contained 15 or more non-consecutive matches (i.e., a maximum simple homology of 15/24 (62.5%) or more were eliminated. This results in a smaller set of candidate sequences from which another sequence is selected that is now referred to as sequence 102. The smaller set of candidate sequences is now compared to sequence 102 eliminating sequences that contained 15 or more non-consecutive matches and the process is repeated until there are no candidate sequences remaining. Also, any sequence selected from the candidate sequences is eliminated if it has 13 or more consecutive matches with any other previously selected candidate sequence.

The additional set of 73tag sequences so obtained (SEQ ID NOs:101 to 173 of Table 1) is composed of sequences that when compared to any of SEQ ID NOs:1 to 100 of Table I have no greater similarity than the sequences of the original 100 sequence tags of Table I. The sequence set as derived from the original family of non cross hybridizing sequences, SEQ ID NOs:1 to 173 of Table 1, are expected to behave with similar hybridization properties to the sequences having SEQ ID NOs:1 to 100 since it is understood that sequence similarity correlates directly with cross hybridization (Southern et al., Nat. Genet.; 21, 5-9: 1999).

The set of 173 24mer oligonucleotides were expanded to include those having SEQ ID NOs:174 to 210 as follows. The 4mers WXYW, XYXW, WXXW, WYYW, XYYX, YXYX, YXXY and XYXY where W=G, X=A, and Y=U/T were used in combination with the fourteen 4mers used in the generation of SEQ ID NOs:1 to 173 to generate potential 24-base oligonucleotides. Excluded from the set were those containing the sequence patterns GG, AAAA and TTTT. To be included in the set of additional 24mers, a sequence also had to have at least one of the 4mers containing two G's: WXYW (GATG), WYXW (GTAG), WXXW (GAAG), WYYW (GTTG) while also containing exactly six G's. Also required for a 24mer to be included was that there be at most six bases between every neighboring pair of G's. Another way of putting this is that there are at most six non-G's between any two G's. Also, each G nearest the 5′-end of its oligonucleotide (the left-hand side as written in Table I) was required to occupy one of the first to seventh positions (counting the 5′-terminal position as the first position.) A set of candidate sequences was obtained by eliminating any new sequence that was found to have a maximum simple homology of 16/24 or more with any of the previous set of 173 oligonucleotides (Table 1, SEQ ID NOs:1 to 0.173). As above, an arbitrary 174^(th) sequence was chosen and candidate sequences eliminated by comparison therewith. In this case the permitted maximum degree of simple homology was 16/24. A second sequence was also eliminated if there were ten consecutive matches between the two (i.e., it was notionally possible to generate a phantom sequence containing a sequence of 10 bases that is identical to a sequence in each of the sequences being compared). A second sequence was also eliminated if it was possible to generate a phantom sequence 20 bases in length or greater.

A property of the polynucleotide sequences shown in Table I is that the maximum block homology between any two sequences is never greater than 66⅔ percent. This is because the computer algorithm by which the sequences were initially generated was designed to prevent such an occurrence. It is within the capability of a person skilled in the art, given the family of sequences of Table I, to modify the sequences, or add other sequences while largely retaining the property of minimal-cross hybridization which the polynucleotides of Table I have been demonstrated to have.

There are 210 polynucleotide sequences given in Table I. Since all 210 of this family of polynucleotides can work with each other as a minimally cross-hybridizing set, then any plurality of polynucleotides that is a subset of the 210 can also act as a minimally cross-hybridizing set of polynucleotides. An application in which, for example, 30 molecules are to be sorted using a family of polynucleotide tags and tag complements could thus use any group of 30 sequences shown in Table I. This is not to say that some subsets may be found in practical sense to be more preferred than others. For example, it may be found that a particular subset is more tolerant of a wider variety of conditions under which hybridization is conducted before the degree of cross-hybridization becomes unacceptable.

It may be desirable to use polynucleotides that are shorter in length than the 24 bases of those in Table I. A family of subsequences (i.e., subframes of the sequences illustrated) based on those contained in Table I having as few as 10 bases per sequence could be chosen, so long as the subsequences are chosen to retain homological properties between any two of the sequences of the family important to their non cross-hybridization.

The selection of sequences using this approach would be amenable to a computerized process. Thus for example, a string of 10 contiguous bases of the first 24mer of Table I could be selected: GATTTGTATTGATTGAGATTAAAG.

A string of contiguous bases from the second 24mer could then be selected and compared for maximum homology against the first chosen sequence: TGATTGTAGTATGTATTGATAAAG

Systematic pairwise comparison could then be carried out to determine if the maximum homology requirement of 66⅔ percent is violated: Alignment Matches          GATTTGTATT 1 ATTGATAAAG          GATTTGTATT 0  ATTGATAAAG          GATTTGTATT 1   ATTGATAAAG          GATTTGTATT 1    ATTGATAAAG          GATTTGTATT 1     ATTGATAAAG          GATTTGTATT 1      ATTGATAAAG          GATTTGTATT 3       ATTGATAAAG          GATTTGTATT 1        ATTGATAAAG          GATTTGTATT 2         ATTGATAAAG          GATTTGTATT 2          ATTGATAAAG          GATTTGTATT 5 (*)           ATTGATAAAG          GATTTGTATT 3            ATTGATAAAG          GATTTGTATT 3             ATTGATAAAG          GATTTGTATT 2              ATTGATAAAG          GATTTGTATT 1               ATTGATAAAG          GATTTGTATT 1                ATTGATAAAG          GATTTGTATT 3                 ATTGATAAAG          GATTTGTATT 1                  ATTGATAAAG          GATTTGTATT 0                   ATTGATAAAG

As can be seen, the maximum homology between the two selected subsequences is 50 percent (5 matches out of the total length of 10), and so these two sequences are compatible with each other.

A 10mer subsequence can be selected from the third 24mer sequence of Table I, and pairwise compared to each of the first two 10mer sequences to determine its compatability therewith, etc. and in this way a family of 10mer sequences developed.

It is within the scope of this invention, to obtain families of sequences containing 11mer, 12mer, 13mer, 14mer, 15mer, 16mer, 17mer, 18mer, 19mer, 20mer, 21mer, 22mer and 23mer sequences by analogy to that shown for 10mer sequences.

It may be desirable to have a family of sequences in which there are sequences greater in length than the 24mer sequences shown in Table I. It is within the capability of a person skilled in the art, given the family of sequences shown in Table I, to obtain such a family of sequences. One possible approach would be to insert into each sequence at one or more locations a nucleotide, non natural base or analogue such that the longer sequence should not have greater similarity than any two of the original non cross hybridizing sequences of Table I and the addition of extra bases to the tag sequences should not result in a major change in the thermodynamic properties of the tag sequences of that set for example the GC content must be maintained between 10%-40% with a variance from the average of 20%. This method of inserting bases could be used to obtain a family of sequences up to 40 bases long.

Given a particular family of sequences that can be used as a family of tags (or tag complements), e.g., those of Table I or Table II, or the combined sequences of these two tables, a skilled person will readily recognize variant families that work equally as well.

Again taking the sequences of Table I for example, every T could be converted to an A and vice versa and no significant change in the cross-hybridization properties would be expected to be observed. This would also be true if every G were converted to a C.

Also, all of the sequences of a family could be taken to be constructed in the 5′-3′ direction, as is the convention, or all of the constructions of sequences could be in the opposition direction (3′-5′).

There are additional modifications that can be carried out. For example, C has not been used in the family of sequences. Substitution of C in place of one or more T's of a particular sequence would yield a sequence that is at least as low in homology with every other sequence of the family as the particular sequence chosen to be modified was. It is thus possible to substitute C in place of one or more T's in any of the sequences shown in Table I. Analogously, substituting of C in place of one or more A's is possible, or substituting C in place of one or T's is possible.

It is preferred that the sequences of a given family are of the same, or roughly the same length. Preferably, all the sequences of a family of sequences of this invention have a length that is within five bases of the base-length of the average of the family. More preferably, all sequences are within four bases of the average base-length. Even more preferably, all or almost all sequences are within three bases of the average base-length of the family. Better still, all or almost all sequences have a length that is within two of the base-length of the average of the family.

It is also possible for a person skilled in the art to derive sets of sequences from the family of sequences that is the subject of this patent and remove sequences that would be expected to have undesirable hybridization properties.

Methods for Synthesis of Oligonucleotide Families

Preferably oligonucleotide sequences of the invention are synthesized directly by standard phosphoramidite synthesis approaches and the like (Caruthers et al, Methods in Enzymology; 154, 287-313: 1987; Lipshutz et al, Nature Genet.; 21, 20-24: 1999; Fodor et al, Science; 251, 763-773: 1991). Alternative chemistries involving non natural bases such as peptide nucleic acids or modified nucleosides that offer advantages in duplex stability may also be used (Hacia et al; Nucleic Acids Res; 27: 4034-4039, 1999; Nguyen et al, Nucleic Acids Res.; 27, 1492-1498: 1999; Weiler et al, Nucleic Acids Res.; 25, 2792-2799:1997). It is also possible to synthesize the oligonucleotide sequences of this invention with alternate nucleotide backbones such as phosphorothioate or phosphoroamidate nucleotides. Methods involving synthesis through the addition of blocks of sequence in a step wise manner may also be employed (Lyttle et al, Biotechniques, 19: 274-280 (1995). Synthesis may be carried out directly on the substrate to be used as a solid phase support for the application or the oligonucleotide can be cleaved from the support for use in solution or coupling to a second support.

Solid Phase Supports

There are several different solid phase supports that can be used with the invention. They include but are not limited to slides, plates, chips, membranes, beads, microparticles and the like. The solid phase supports can also vary in the materials that they are composed of including plastic, glass, silicon, nylon, polystyrene, silica gel, latex and the like. The surface of the support is coated with the complementary sequence of the same.

In preferred embodiments, the family of tag complement sequences are derivatized to allow binding to a solid support. Many methods of derivatizing a nucleic acid for binding to a solid support are known in the art (Hermanson G., Bioconjugate Techniques; Acad. Press: 1996). The sequence tag may be bound to a solid support through covalent or non-covalent bonds (Iannone et al, Cytometry; 39: 131-140, 2000; Matson et al, Anal. Biochem.; 224: 110-106, 1995; Proudnikov et al, Anal Biochem; 259: 34-41, 1998; Zammatteo et al, Analytical Biochemistry; 280:143-150, 2000). The sequence tag can be conveniently derivatized for binding to a solid support by incorporating modified nucleic acids in the terminal 5′ or 3′ locations.

A variety of moieties useful for binding to a solid support (e.g., biotin, antibodies, and the like), and methods for attaching them to nucleic acids, are known in the art. For example, an amine-modified nucleic acid base (available from, eg., Glen Research) may be attached to a solid support (for example, Covalink-NH, a polystyrene surface grafted with secondary amino groups, available from Nunc) through a bifunctional crosslinker (e.g., bis(sulfosuccinimidyl suberate), available from Pierce). Additional spacing moieties can be added to reduce steric hindrance between the capture moiety and the surface of the solid support.

Attaching Tags to Analytes for Sorting

A family of oligoucleotide tag sequences can be conjugated to a population of analytes most preferably polynucleotide sequences in several different ways including but not limited to direct chemical synthesis, chemical coupling, ligation, amplification, and the like. Sequence tags that have been synthesized with primer sequences can be used for enzymatic extension of the primer on the target for example in PCR amplification.

Detection of Single Nucleotide Polymorphisms Using Primer Extension

There are a number of areas of genetic analysis where families of non cross hybridizing sequences can be applied including disease dagnosis, single nucleotide polymorphism analysis, genotyping, expression analysis and the like. One such approach for genetic analysis referred to as the primer extension method (also known as Genetic Bit Analysis (Nikiforov et al, Nucleic Acids Res.; 22, 4167-4175: 1994; Head et al Nucleic Acids Res.; 25, 5065-5071: 1997)) is an extremely accurate method for identification of the nucleotide located at a specific polymorphic site within genomic DNA. In standard primer extension reactions, a portion of genomic DNA containing a defined polymorphic site is amplified by PCR using primers that flank the polymorphic site. In order to identify which nucleotide is present at the polymorphic site, a third primer is synthesized such that the polymorphic position is located immediately 3′ to the primer. A primer extension reaction is set up containing the amplified DNA, the primer for extension, up to 4 dideoxynucleoside triphosphates, each labelled with a different fluorescent dye and a DNA polymerase such as the Klenow subunit of DNA Polymerase 1. The use of dideoxy nucleotides ensure that a single base is added to the 3′ end of the primer, a site corresponding to the polymorphic site. In this way the identity of the nucleotide present at a specific polymorphic site can be determined by the identity of the fluorescent dye-labelled nucleotide that is incorporated in each reaction. One major drawback to this approach is its low throughput. Each primer extension reaction is carried out independently in a separate tube.

Universal sequences can be used to enhance the throughput of primer extension assay as follows. A region of genomic DNA containing multiple polymorphic sites is amplified by PCR. Alternately, several genomic regions containing one or more polymorphic sites each are amplified together in a multiplexed PCR reaction. The primer extension reaction is carried out as described above except that the primers used are chimeric, each containing a unique universal tag at the 5′ end and the sequence for extension at the 3′ end. In this way, each gene-specific sequence would be associated with a specific universal sequence. The chimeric primers would be hybridized to the amplified DNA and primer extension carried out as described above. This would result in a mixed pool of extended primers, each with a specific fluorescent dye characteristic of the incorporated nucleotide. Following the primer extension reaction, the mixed extension reactions are hybridized to an array containing probes that are reverse complements of the universal sequences on the primers. This would segregate the products of a number of primer extension reactions into discrete spots. The fluorescent dye present at each spot would then identify the nucleotide incorporated at each specific location.

Kits Using Families of Tag Sequences

The families of non cross-hybridizing sequences may be provided in kits for use in for example genetic analysis. Such kits include at least one set of non cross hybridizing sequences in solution or on a solid support. Preferably the sequences are attached to microparticles and are provided with buffers and reagents that are appropriate for the application. Reagents may include enzymes, nucleotides, fluorescent labels and the like that would be required for specific applications. Instructions for correct use of the kit for a given application will be provided.

EXAMPLES Example 1

Demonstrate Non Cross Talk Behavior

One hundred oligonucleotide probes corresponding to a family of non-cross talking oligonucleotides from Table I were synthesized by Integrated DNA Technologies (IDT, Coralville Iowa). These oligonucleotides incorporated a C₆ aminolink group coupled to the 5′ end of the oligo through a C₁₈ ethylene glycol spacer. These probes were used to prepare microarrays as follows. The probes were resuspended at a concentration of 50 μM in 150 mM NaPO4, pH 8.5. The probes were spotted onto the surface of a SuperAldehyde slide (Telechem Int., Sunnyvale Calif.) using and SDDC-II microarray spotter (ESI, Toronto Ont). The spots formed were approximately 120 FM in diameter with 200 μM centre-to-centre spacing. Each probe was spotted 8 times on each microarray. Following spotting, the arrays were processed essentially as described by the slide manufacturer. Briefly, the arrays were treated with 67 mM sodium borohydride in PBS/EtOH (3:1) for 5 minutes then washed with 4 changes of 0.1% SDS. The arrays were not boiled.

One hundred labelled oligonucleotide targets were also synthesized by IDT. The sequence of these targets corresponded to the reverse complement of the 100 probe sequences. The targets were labelled at the 5′ end with Cy3.

Each Cy3-labeled target oligonucleotide was hybridized separately to two microarrays each of which contained all 100 oligonucleotide probes. Hybridizations were carried out at 42° C. for 2 hours in a 40 ll reaction and contained 40 nM of the labelled target suspended in 10 mM Tris HCl, pH 8.3, 50 mM KCl, 0.1% Tween 20. These are low stringency hybridization conditions designed to provide a rigorous test of the performance of the family of non-cross hybridizing sequences. Hybridizations were carried out by depositing the hybridization solution on a clean cover slip then carefully positioning the microarray slide over the cover slip in order to avoid bubbles. The slide was then inverted and transferred to a humid chamber for incubation. Following hybridization, the cover slip was removed and the microarray was washed in hybridization buffer for 15 minutes at room temperature. The slide was then dried by brief centrifugation.

Hybridized microarrays were scanned using a ScanArray Lite (GSI-Lumonics, Billerica Mass.). The laser power and photomultiplier tube voltage used for scanning each hybridized microarray were optimized in order to maximize the signal intensity from the spots representing the perfect match.

The results of a sample hybridization are shown in FIG. 1. FIG. 1 a shows the hybridization pattern seen when a microarray containing all 100 probes was hybridized with the target complementary to probe 181234. The 4 sets of paired spots correspond to the probe complementary to the target. FIG. 1 b shows the pattern seen when a similar array was hybridized with a mix of all 100 targets.

Example 2 Tag Sequences Used in Sorting Polynucleotides

The family of non cross hybridizing sequence tags or a subset thereof can be attached to oligonucleotide probe sequences during synthesis and used to generate amplified probe sequences. In order to test the feasibility of PCR amplification with non cross hybridizing sequence tags and subsequently addressing each respective sequence to its appropriate location on two-dimensional or bead arrays, the following experiment was devised. A 24mer tag sequence was connected in a 5′-3′ specific manner to a p53 exon specific sequence (20mer reverse primer). The connecting p53 sequence represented the inverse complement of the nucleotide gene sequence. To facilitate the subsequent generation of single stranded DNA post-amplification the tag-Reverse primer was synthesized with a phosphate modification (PO₄) on the 5′-end. A second PCR primer was also generated for each desired exon, which represented the Forward (5′-3′) amplification primer. In this instance the Forward primer was labeled with a 5′-biotin modification to allow detection with Cy3-avidin or equivalent.

A practical example of the aforementioned description is as follows: For exon 1 of the human p53 tumor suppressor gene sequence the following tag-Reverse primer was generated:                           222087                       222063 5′-P04-GATTGTAAGATTTGATAAAGTGTA-TCCAGGGAAGCGTGTCACCGTCGT-3′       Tag Sequence # 3                     Exon 1 Reverse

The numbering above the Exon-1 reverse primer represents the genomic nucleotide positions of the indicated bases. The corresponding Exon-1 Forward primer sequence is as follows:           221873                      221896 5′-Biotin-TCATGGCGACTGTCCAGCTTTGTG-3′

In combination these primers will amplify a product of 214 bp plus a 24 bp tag extension yielding a total size of 0.238 bp. Once amplified, the PCR product was purified using a QIAquick PCR purification kit and the resulting DNA was quantified. To generate single stranded DNA the DNA was subjected to—exonuclease digestion thereby resulting in the exposure of a single stranded sequence (anti-tag) complementary to the tag-sequence covalently attached to the solid phase array. The resulting product was heated to 95° C. for 5 minutes and then directly applied to the array at a concentration of 10-50 nM. Following hybridization and concurrent sorting, the tag-Exon 1 sequences were visualized using Cy3-streptavidin. In addition to direct visualization of the biotinylated product, the product itself can now act as a substrate for further analysis of the amplified region, such as SNP detection and haplotype determination.

The present invention also includes a family of 1168 24mer polynucleotides that have been demonstrated to be minimally cross-hybridizing with each other. This family of polynucleotides is thus useful as a family of tags, and their complements as tag complements.

In order to be considered for inclusion into the family, a sequence had to satisfy a certain number of rules regarding its composition. For example, repetitive regions that present potential hybridization problems such as four or more of a similar base (e.g., AAAA or TTTT) or pairs of Gs were forbidden. Another rule is that each sequence contains exactly six Gs and no Cs, in order to have sequences that are more or less isothermal. Also required for a 24mer to be included is that there must be at most six bases between every neighboring pair of Gs. Another way of putting this is that there are at most six non-Gs between any two consecutive Gs. Also, each G nearest the 5′-end (resp. 3′-end) of its oligonucleotide (the left-hand (resp. right-hand) side as written in Table II) was required to occupy one of the first to seventh positions (counting the 5′-terminal (resp. 3′-terminal) position as the first position.)

The process used to design families of sequences that do not exhibit cross-hybridization behavior is illustrated generally in FIG. 5). Depending on the application for which these families of sequences will be used, various rules are designed. A certain number of rules can specify constraints for sequence composition (such as the ones described in the previous paragraph). The other rules are used to judge whether two sequences are too similar. Based on these rules, a computer program can derive families of sequences that exhibit minimal or no cross-hybridization behavior. The exact method used by the computer program is not crucial since various computer programs can derive similar families based on these rules. Such a program is for example described in international patent application No. PCT/CA 01/00141 published under WO 01/59151 on Aug. 16, 2001. Other, programs can use different methods, such as the ones summarized below.

A first method of generating a maximum number of minimally cross-hybridizing polynucleotide sequences starts with any number of non-cross-hybridizing sequences, for example just one sequence, and increases the family as follows. A certain number of sequences is generated and compared to the sequences already in the family. The generated sequences that exhibit too much similarity with sequences already in the family are dropped. Among the “candidate sequences” that remain, one sequence is selected and added to the family. The other candidate sequences are then compared to the selected sequence, and the ones that show too much similarity are-dropped. A new sequence is selected from the remaining candidate sequences, if any, and added to the family, and soon until there are no candidate sequences left. At this stage, the process can be repeated (generating a certain number of sequences and comparing them to the sequences in the family, etc.) as often as desired. The family obtained at the end of this method contains only minimally cross-hybridizing sequences.

A second method of generating a maximum number of minimally cross-hybridizing polynucleotide sequences starts with a fixed-size family of polynucleotide sequences. The sequences of this family can be generated randomly or designed by some other method. Many sequences in this family may not be compatible with each other, because they show too much similarity and are not minimally cross-hybridizing. Therefore, some sequences need to be replaced by new ones, with less similarity. One way to achieve this consists of repeatedly replacing a sequence of the family by the best (that is, lowest similarity) sequence among a certain number of (for example, randomly generated) sequences that are not part of the family. This process can be repeated until the family of sequences shows minimal similarity, hence minimal cross-hybridizing, or until a set number of replacements has occurred. If, at the end of the process, some sequences do not obey the similarity rules that have been set, they can be taken out of the family, thus providing a somewhat smaller family that only contains minimally cross-hybridizing sequences. Some additional rules can be added to this method in order to make it more efficient, such as rules to determine which sequence will be replaced.

Such methods have been used to obtain the 1168 non-cross-hybridizing tags of Table II that are also the subject of this patent application.

One embodiment of the invention is a composition comprising molecules for use as tags or tag complements wherein each molecule comprises an oligonucleotide selected from a set of oligonucleotides based on the group of sequences set out in Table IIA, wherein each of the numeric identifiers 1 to 3-(see the Table) is a nucleotide base selected to be different from the others of 1 to 3. According to this embodiment, several different families of specific sets of oligonucleotide sequences are described, depending upon the assignment of bases made to the numeric identifiers 1 to 3.

The sequences contained in Table II have a mathematical relationship to each other, described as follows.

Let S and T be two DNA sequences of lengths s and t respectively. While the term “alignment” of nucleotide sequences is widely used in the field of biotechnology, in the context of this invention the term has a specific meaning illustrated here. An alignment of S and T is a 2xp matrix A (with p 2 s and p≧t) such that the first (or second) row of A contains the characters of S (or T respectively) in order, interspersed with p−s (or p−t respectively) spaces. It assumed that no column of the alignment matrix contains two spaces, i.e., that any alignment in which a column contains two spaces is ignored and not considered here. The columns containing the same base in both rows are called matches, while the columns containing different bases are called mismatches. Each column of an alignment containing a space in its first row is called an insertion and each column containing a space in its second row is called a deletion while a column of the alignment containing a space in either row is called an indel. Insertions and deletions within a sequence are represented by the character ‘-’. A gap is a continuous sequence of spaces in one of the rows (that is neither immediately preceded nor immediately followed by another space in the same row), and the length of a gap is the number of spaces in that gap. An internal gap is one in which its first space is preceded by a base and its last space is followed by a base and an internal indel is an belonging to an internal gap. Finally, a block is a continuous sequence of matches (that is neither immediately preceded nor immediately followed by another match), and the length of a block is the number of matches in that block. In order to illustrate these definitions, two sequences S=TGATCGTAGCTACGCCGCG (of length s=19; SEQ ID NO:1169) and T=CGTACGATTGCAACGT (of length t=16, SEQ ID NO:1170) are considered. Exemplary alignment R₁ of S and T (with p=23) is: Alignment R₁: — — — — T G A T C G T A G C T A C G C C G C G C G T A C G A T — — T — G C A A C G T — — — — 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Columns 1 to 4, 9, 10, 12 and 20 to 23 are indels, columns 6, 7, 8, 11, 13, 14, 16, 17 and 18 are matches, and columns 5, 15 and 19 are mismatches. Columns 9 and 10 form a gap of length 2, while columns 16 to 18 form a block of length 3. Columns 9, 10 and 12 are internal indels.

A score is assigned to the alignment A of two sequences by assigning weights to each of matches, mismatches and gaps as follows:

-   -   the reward for a match m,     -   the penalty for a mismatch mm,     -   the penalty for opening a gap og     -   the penalty for extending a gap eg.         Once these values are set, a score to each column of the         alignment is assigned according to the following rules:     -   1. assign 0 to each column preceding the first match and to each         column following the last match.     -   2. for each of the remaining columns, assign m if it is a match,         mm if it is a mismatch, -og-eg if it is the first indel of a         gap, -eg if it is an indel but not the first indel of a gap.         The score of the alignment A is the sum of the scores of its         columns. An alignment is said to be of maximum score if no other         alignment of the same two sequences has a higher score (with the         same values of m, mm, og and eg). A person knowledgeable in the         field will recognize this method of scoring an alignment as         scoring a local (as opposed to global) alignment with affine gap         penalties (that is, gap penalties that can distinguish between         the first indel of a gap and the other indels). It will be         appreciated that the total number of indels that open a gap is         the same as the total number of gaps and that an internal indel         is not one of those assigned a 0 in rule (1) above. It will also         be noted that foregoing rule (1) assigns a 0 for non-internal         mismatches. An internal mismatch is a mismatch that is preceded         and followed (not necessarily immediately) by a match.

As an illustration, if the values of m, mm, og and eg are set to 3, 1, 2 and 1 respectively, alignment R₁ has a score of. 19, determined as shown below: Scoring of Alignment R₁ — — — — T G A T C G T A G C T A C G C C G C G C G T A C G A T — — T — G C A A C G T — — — — 0 0 0 0 0 3 3 3 −3 −1 3 −3 3 3 −1 3 3 3 0 0 0 0 0 Note that for two given sequences S and T, there are numerous alignments. There are often several alignments of maximum score.

Based on these alignments, five sequence similarity measures are defined as follows. For two sequences S and T, and weights {m, mm, og, eg}:

-   -   M1 is the maximum number of matches over all alignments free of         internal indels;     -   M2 is the maximum length of a block over all alignments;     -   M3 is the maximum number of matches over all alignments of         maximum score;     -   M4 is the maximum sum of the lengths of the longest two blocks         over all alignments of maximum score;     -   M5 is the maximum sum of the lengths of all the blocks of length         at least 3, over all alignments of maximum score.         Notice that, by definition, the following inequalities between         these similarity measures are obtained: M4≦M3 and M5≦M3. Also,         in order to determine M2 it is sufficient to determine the         maximum length of a block over all alignments free of internal         indels. For two given sequences, the values of M3 to M5 can vary         depending on the values of the weights {m, mm, og, eg}, but not         M1 and M2.

For weights {3, 1, 2, 1}, the illustrated alignment is not a maximum score alignment of the two example sequences. But for weights {6, 6, 0, 6} it is; hence this alignment shows that for these two example sequences, and weights {6, 6, 0, 6}, M2≧3, M3≧9, M4≧6 and M5≧6. In order to determine the exact values of M1 to M5, all the necessary alignments need to be considered. M1 add M2 can be found by looking at the s+t−1 alignments free of internal indels, where s and t are the lengths of the two sequences considered. Mathematical tools known as dynamic programming can be implemented on a computer and used to determine M3 to M5 in a very quick way. Using a computer program to do these calculations, it was determined that:

-   -   with the weights {6, 6, 0, 6}, M1=8, M2=4, M3=10, M4=6 and M5=6;     -   with the weights {3, 1, 2, 1}, M1=8, M2=4, M3=10, M4=6 and M5=4.         According to the preferred embodiment of this invention, two         sequences S and T each of length 24 are too similar if at least         one of the following happens:     -   M1>16 or     -   M2>13 or     -   M3>20 or     -   M4>16 or     -   M5>19         when using either weights {6, 6, 0, 6}, or {6, 6, 5, 1}, or {6,         2, 5, 1}, or {6, 6, 6, 0}. In other words, the five similarity         measures between S and T are determined for each of the above         four sets of weights, and checked against these thresholds (for         a total of 20 tests).

The above thresholds of 16, 13, 20, 16 and 19, and the above sets of weights, were used to obtain the sequences listed in Table I. Additional sequences can thus be added to those of Table I as long as the above alignment rules are obeyed for all sequences.

It is also possible to alter thresholds M1, M2, etc., while remaining within the scope of this invention. It is thus possible to substitute or add sequences to those of Table II, or more generally to those of Table IIA to obtain other sets of sequences that would also exhibit reasonably low cross-hybridization. More specifically, a set of 24mer sequences in which there are no two sequences that are too similar, where too similar is defined as:

-   -   M1>19 or     -   M2>17 or     -   M3>21 or     -   M4>18 or     -   M5>20         when using either weights {6, 6, 0, 6}, or {6, 6, 5, 1}, or {6,         2, 5, 1}, or {6, 6, 6, 0}, would also exhibit low         cross-hybridization. Reducing any of the threshold values         provides sets of sequences with even lower cross-hybridization.         Alternatively, ‘too similar’ can also be defined as:     -   M1>19 or     -   M2>17 or     -   M3>21 or     -   M4>18 or     -   M5>20         when using either weights {3, 1, 2, 1}. Alternatively, other         combinations of weights will lead to sets of sequences with low         cross-hybridization.

Notice that using weights {6, 6, 0, 6} is equivalent to using weights {1, 1, 0, 1}, or weights {2, 2, 0, 2}, . . . (that is, for any two sequences, the values of M1 to M5 are exactly the same whether weights {6, 6, 0, 6} or {1, 1, 0, 1} or {2, 2, 0, 2} or any other multiple of {1, 1, 0, 1} is used).

When dealing with sequences of length other than 24, or sequences of various lengths, the definition of similarity can be adjusted. Such adjustments are obvious to the persons skilled in the art. For example, when comparing a sequence of length L1 with a sequence of length L2 (with L1<L2), they can be considered as too similar when

-   M1>19/24×L1 -   M2>17/24×L1 -   M3>21/24×L1 -   M4>18/24×L1 -   M5>20/24×L1     when using either weights {6, 6, 0, 6}, or {6, 6, 5, 1}, or {6, 2,     5, 1} or {6, 6, 6, 0}.

Polynucleotide sequences can be composed of a subset of natural bases most preferably A, T and G. Sequences that are deficient in one base possess useful characteristics, for example, in reducing potential secondary structure formation or reduced potential for cross hybridization with nucleic acids in nature. Also, it is preferable to have tag sequences that behave isothermally. This can be achieved for example by maintaining a constant base composition for all sequences such as six Gs and eighteen As or Ts' for each sequence. Additional sets of sequences can be designed by extrapolating on the original family of non-cross-hybridizing sequences by simple methods known to those skilled in the art.

In order to validate the sequence set, a subset of sequences from the family of 1168 sequence tags was selected and characterized, in terms of the ability of these sequences to form specific duplex structures with their complementary sequences, and the potential for cross-hybridization within the sequence set. See Example 4, below. The subset of 100 sequences was randomly selected, and analyzed using the Luminex¹⁰⁰ LabMAP™ platform. The 100 sequences were chemically immobilized onto the set of 100 different Luminex microsphere populations, such that each specific sequence was coupled to one spectrally distinct microsphere population. The pool of 100 microsphere-immobilized probes was then hybridized with each of the 100 corresponding complementary sequences. Each sequence was examined individually for its specific hybridization with its complementary sequence, as well as for its non-specific hybridization with the other 99 sequences present in the reaction. This analysis demonstrated the propensity of each sequence to hybridize only to its complement (perfect match), and not to cross-hybridize appreciably with any of the other oligonucleotides present in the hybridization reaction.

It is within the capability of a person skilled in the art, given the family of sequences of Table II, to modify the sequences, or add other sequences while largely retaining the property of minimal cross-hybridization which the polynucleotides of Table II have been demonstrated to have.

There are 1168 polynucleotide sequences given in Table II. Since all 1168 of this family of polynucleotides can work with each other as a minimally cross-hybridizing set, then any plurality of polynucleotides that is a subset of the 1168 can also act as a minimally cross-hybridizing set of polynucleotides. An application in which, for example, 30 molecules are to be sorted using a family of polynucleotide tags and tag complements could thus use any group of 30 sequences shown in Table II. This is not to say that some subsets may be found in a practical sense to be more preferred than others. For example, it may be found that a particular subset is more tolerant of a wider variety of conditions under which hybridization is conducted before the degree of cross-hybridization becomes unacceptable.

It may be desirable to use polynucleotides that are shorter in length than the 24 bases of those in Table II. A family of subsequences (i.e., subframes of the sequences illustrated) based on those contained in Table II having as few as 10 bases per sequence could be chosen, so long as the subsequences are chosen to retain homological properties between any two of the sequences of the family important to their non cross-hybridization.

The selection of sequences using this approach would be amenable to a computerized process. Thus for example, a string of 10 contiguous bases of the first 24mer of Table II could be selected: AAATTGTGAAAGATTGTTTGTGTA (SEQ ID NO:1).

The same string of contiguous bases from the second 24mer could then be selected and compared for similarity against the first chosen sequence: GTTAGAGTTAATTGTATTTGATGA (SEQ ID NO:2 of Table II). A systematic pairwise comparison could then be carried out to determine if the similarity requirements are violated. If the pair of sequences does not violate any set property, a 10mer subsequence can be selected from the third 24mer sequence of Table II, and compared to each of the first two 10mer sequences (in a pairwise fashion to determine its compatibility therewith, etc. In this way a family of 10mer sequences may be developed.

It is within the scope of this invention, to obtain families of sequences containing 1mer, 12mer, 13mer, 14mer, 15mer, 16mer, 17mer, 18mer, 19mer, 20mer, 21mer, 22mer and 23mer sequences by analogy to that shown for 10mer sequences. It may be desirable to have a family of sequences in which there are sequences greater in length than the 24mer sequences shown in Table II. It is within the capability of a person skilled in the art, given the family of sequences shown in Table II, to obtain such a family of sequences. One possible approach would be to insert into each sequence at one or more locations a nucleotide, non-natural base or analogue such that the longer sequence should not have greater similarity than any two of the original non-cross-hybridizing sequences of Table II and the addition of extra bases to the tag sequences should not result in a major change in the thermodynamic properties of the tag sequences of that set for example the GC content must be maintained between 10%-40% with a variance from the average of 20%. This method of inserting bases could be used to obtain, for example, a family of sequences up to 40 bases long.

Given a particular family of sequences that can be used as a family of tags (or tag complements), e.g., those of Table II, a skilled person will readily recognize variant families that work equally as well.

Again taking the sequences of Table II for example, every T could be converted to an A and vice versa and no significant change in the cross-hybridization properties would be expected to be observed. This would also be true if every G were converted to a C.

Also, all of the sequences of a family could be taken to be constructed in the 5′-3′ direction, as is the convention, or all of the constructions of sequences could be in the opposition direction (3′-5′).

There are additional modifications that can be carried out. For example, C has not been used in the family of sequences. Substitution of C in place of one or more G's of a particular sequence would yield a sequence that is at least as low in homology with every other sequence of the family as was the particular sequence chosen for modification. It is thus possible to substitute C in place of one or more G's in any of the sequences shown in Table II. Analogously, substituting of C in place of one or more A's is possible, or substituting C in place of one or T's is possible.

It is preferred that the sequences of a given family are of the same, or roughly the same length. Preferably, all the sequences of a family of sequences of this invention have a length that is within five bases of the base-length of the average of the family. More preferably, all sequences are within four bases of the average base-length. Even more preferably, all or almost all sequences are within three bases of the average base-length of the family. Better still, all or almost all sequences have a length that is within two of the base-length of the average of the family, and even better still, within one of the base-length of the average of the family.

It is also possible for a person skilled in the art to derive sets of sequences from the family of sequences described in this specification and remove sequences that would be expected to have undesirable hybridization properties.

Example 3 Cross Talk Behavior of Sequence on Beads

A group of 100 of the sequences of Table I was tested for feasibility for use as a family of minimally cross-hybridizing oligonucleotides. The 100 sequences selected are separately indicated in Table I along with the numbers assigned to the sequences in the tests.

The tests were conducted using the Luminex LabMAP™ platform available from Luminex Corporation, Austin, Tex., U.S.A. The one hundred sequences used as probes; were synthesized as oligonucleotides by Integrated DNA Technologies (IDT, Coralville, Iowa, U.S.A.). Each probe included a C₆ aminolink group coupled to the 5′-end of the oligonucleotide through a C₁₂ ethylene glycol spacer. The C₆ aminolink molecule is a six carbon spacer containing an amine group that can be used for attaching the oligonucleotide to a solid support. One hundred oligonucleotide targets (probe complements), the sequence of each being the reverse complement of the 100 probe sequences, were also synthesized by IDT. Each target was labelled at its 5′-end with biotin. All oligonucleotides were purified using standard desalting procedures, and were reconstituted to a concentration of approximately 200 μM in sterile, distilled water for use. Oligonucleotide concentrations were determined spectrophotometrically using extinction coefficients provided by the supplier.

Each probe was coupled by its amino linking group to a carboxylated fluorescent microsphere of the LabMAP system according to the Luminex¹⁰⁰ protocol. The microsphere, or bead, for each probe sequence has unique, or spectrally distinct, light absorption characteristics which permits each probe to be distinguished from the other probes. Stock bead pellets were dispersed by sonication and then vortexing. For each bead population, approximately five million microspheres (400 μL) were removed from the stock tube using barrier tips and added to a 1.5 mL Eppendorf tube (USA Scientific). The microspheres were then centrifuged, the supernatant was removed, and beads were resuspended in 25 μL of 0.2 M MES (2-(N-morpholino)ethane sulfonic acid) (Sigma), pH 4.5, followed by vortexing and sonication. One nmol of each probe (in a 25 μL volume) was added to its corresponding bead population. A volume of 2.5 μL of EDC cross-linker (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (Pierce), prepared immediately before use by adding 1.0 mL of sterile ddH₂O to 10 mg of EDC powder, was added to each microsphere population. Bead mixes were then incubated for 30 minutes at room temperature in the dark with periodic vortexing. A second 2.5 μL aliquot of freshly prepared EDC solution was then added followed by an additional 30 minute incubation in the dark. Following the second EDC incubation, 1.0 mL of 0.02% Tween-20 (BioShop) was added to each bead mix and vortexed. The microspheres were centrifuged, the supernatant was removed, and the beads were resuspended in 1.0 mL of 0.1% sodium dodecyl sulfate (Sigma). The beads were centrifuged again and the supernatant removed. The coupled beads were resuspended in 100 μL of 0.1 M MES pH 4.5. Bead concentrations were then determined by diluting each preparation 100-fold in ddH₂O and enumerating using a Neubauer BrightLine Hemacytometer. Coupled beads were stored as individual populations at 2-8° C. protected from light.

The relative oligonucleotide probe density on each bead population was assessed by Terminal Deoxynucleotidyl Transferase (TdT) end-labelling with biotin-ddUTPs. TdT was used to label the 3′-ends of single-stranded DNA with a labeled ddNTP. Briefly, 180 μL of the pool of 100 bead populations (equivalent to about 4000 of each bead type) to be used for hybridizations was pipetted into an Eppendorf tube and centrifuged. The supernatant was removed, and the beads were washed in 1×TdT buffer. The beads were then incubated with a labelling reaction mixture, which consisted of 5×TdT buffer, 25 mM CoCl₂, and 1000 pmol of biotin-16-ddUTP (all reagents were purchased from Roche). The total reaction volume was brought up to 85.5 μL with sterile, distilled H₂O, and the samples were incubated in the dark for 1 hour at 37° C. A second aliquot of enzyme was added, followed by a second 1 hour incubation. Samples were run in duplicate, as was the negative control, which contained all components except the TdT. In order to remove unincorporated biotin-ddUTP, the beads were washed 3 times with 200 μL of hybridization buffer, and the beads were resuspended in 50 μL of hybridization buffer following the final wash. The biotin label was detected spectrophotometrically using SA-PE (streptavidin-phycoerythrin conjugate). The streptavidin binds to biotin and the phycoerythrin is spectrally distinct from the probe beads. The 10 mg/mL stock of SA-PE was diluted 100-fold in hybridization buffer, and 15 μL of the diluted SA-PE was added directly to each reaction and incubated for 15 minutes at 37° Celsius. The reactions were analyzed on the Luminex¹⁰⁰ LabMAP. Acquisition parameters were set to measure 100 events per bead using a sample volume of 50 μL.

The results obtained are shown in FIG. 2. As can be seen the Mean Fluorescent Intensity (MFI) of the beads varies from 277.75 to 2291.08, a range of 8.25-fold. Assuming that the labelling reactions are complete for all of the oligonucleotides, this illustrates the signal intensity that would be obtained for each type of bead at this concentration if the target (i.e., labelled complement) was bound to the probe sequence to the full extent possible.

The cross-hybridization of targets to probes was evaluated as follows. 100 oligonucleotide probes linked to 100 different bead populations, as described above, were combined to generate a master bead mix, enabling multiplexed reactions to be carried out. The pool of microsphere-immobilized probes was then hybridized individually with each biotinylated target. Thus, each target was examined individually for its specific hybridization with its complementary bead-immobilized sequence, as well as for its non-specific hybridization with the other 99 bead-immobilized universal sequences present in the reaction. For each hybridization reaction, 25 μL bead mix (containing about 2500 of each bead population in hybridization buffer) was added to each well of a 96-well Thermowell PCR plate and equilibrated at 37° C. Each target was diluted to a final concentration of 0.002 fmol/μL in hybridization buffer, and 25 μL (50 fmol) was added to each well, giving a final reaction volume of 50 μL. Hybridization buffer consisted of 0.2 M NaCl, 0.1 M Tris, 0.08% Triton X-100, pH 8.0 and hybridizations were performed at 37° C. for 30 minutes. Each target was analyzed in triplicate and six background samples (i.e. no target) were included in each plate. A SA-PE conjugate was used as a reporter, as described above. The 10 mg/mL stock of SA-PE was diluted 100-fold in hybridization buffer, and 15 μL of the diluted SA-PE was added directly to each reaction, without removal of unbound target, and incubated for 15 minutes at 37° C. Finally, an additional 35 μL of hybridization buffer was added to each well, resulting in a final volume of 100 μL per well prior to analysis on the Luminex¹⁰⁰ LabMAP. Acquisition parameters were set to measure 100 events per bead using a sample volume of 80 μL.

The percent hybridization was calculated for any event in which the NET MFI was at least 3 times the zero target background. In other words, a calculation was made for any sample where (MFI_(sample)−MFI_(zero target background))/MFI_(zero target background)≧3.

A “positive” cross-talk event (i.e., significant mismatch or cross-hybridization) was defined as any event in which the net median fluorescent intensity (MFI_(sample)−MFI_(zero target background)) generated by a mismatched hybrid was greater than or equal to the arbitrarily set limit of 10% that of the perfectly matched hybrid determined under identical conditions. As there are 100 probes and 100 targets, there are 100×100=10,0000 possible different interactions possible of which 100 are the result of perfect hybridizations. The remaining 9900 result from hybridization of a target with a mismatched probe.

The results obtained are illustrated in FIG. 3. The ability of each target to be specifically recognized by its matching probe is shown of the possible 9900 non-specific hybridization events that could have occurred when the 100 targets were each exposed to the pool of 100 probes, 6 events were observed. Of these 6 events, the highest non-specific event generated a signal equivalent to 10.2% of the signal observed for the perfectly matched pair (i.e. specific hybridization event).

Each of the 100 targets was thus examined individually for specific hybridization with its complement sequence as incorporated onto a microsphere, as well as for non-specific hybridization with the complements of the other 99 target sequences. Representative hybridization results for target 16 (complement of probe -16, Table I) are shown in FIG. 4. Probe 16 was found to hybridize only to its perfectly-matched target. No cross-hybridization with any of the other 99 targets was observed.

The foregoing results demonstrate the possibility of incorporating the 210 sequences of Table I, or any subset thereof, into a multiplexed system with the expectation that most if not all sequences can be distinguished from the others by hybridization. That is, it is possible to distinguish each target from the other targets by hybridization of the target with its precise complement and minimal hybridization with complements of the other targets.

Methods for Synthesis of Oligonucleotide Families

Preferably oligonucleotide sequences of the invention are synthesized directly by standard phosphoramidite synthesis approaches and the like (Caruthers et al, Methods in Enzymology; 154, 287-313: 1987; Lipshutz et al, Nature Genet.; 21, 20-24: 1999; Fodor et al, Science; 251, 763-773: 1991). Alternative chemistries involving non natural bases such as peptide nucleic acids or modified nucleosides that offer advantages in duplex stability may also be used (Hacia et al; Nucleic Acids Res; 27: 4034-4039, 1999; Nguyen et al, Nucleic Acids Res.; 27, 1492-1498: 1999; Weiler et al, Nucleic Acids Res.; 25, 2792-2799:1997). It is also possible to synthesize the oligonucleotide sequences of this invention with alternate nucleotide backbones such as phosphorothioate or phosphoroamidate nucleotides. Methods involving synthesis through the addition of blocks of sequence in a stepwise-manner may also be employed (Lyttle et al, Biotechniques, 19: 274-280 (1995). Synthesis may be carried out directly on the substrate to be used as a solid phase support for the application or the oligonucleotide can be cleaved from the support for use in solution or coupling to a second support.

Solid Phase Supports

There are several different solid phase supports that can be used with the invention. They include but are not limited to slides, plates, chips, membranes, beads, microparticles and the like. The solid phase supports can also vary in the materials that they are composed of including plastic, glass, silicon, nylon, polystyrene, silica gel, latex and the like. The surface of the support is coated with the complementary tag sequences by any conventional means of attachment.

In preferred embodiments, the family of tag complement sequences is derivatized to allow binding to a solid support. Many methods of derivatizing a nucleic acid for binding to a solid support are known in the art (Hermanson G., Bioconjugate Techniques; Acad. Press: 1996). The sequence tag may be bound to a solid support through covalent or non-covalent bonds (Iannone et al, Cytometry; 39: 131-140, 2000; Matson et al, Anal. Biochem.; 224: 110-106, 1995; Proudnikov et al, Anal Biochem; 259: 34-41, 1998; Zammatteo et al, Analytical Biochemistry; 280:143-150, 2000). The sequence tag can be conveniently derivatized for binding to a solid support by incorporating modified nucleic acids in the terminal 5′ or 3′ locations.

A variety of moieties useful for binding to a solid support (e.g., biotin, antibodies, and the like), and methods for attaching them to nucleic acids, are known in the art. For example, an amine-modified nucleic acid base (available from, eg., Glen Research) may be attached to a solid support (for example, Covalink-NH, a polystyrene surface grafted with secondary amino groups, available from Nunc) through a bifunctional crosslinker (e.g., bis(sulfosuccinimidyl suberate), available from Pierce). Additional spacing moieties can be added to reduce steric hindrance between the capture moiety and the surface of the solid support.

Attaching Tags to Analytes for Sorting

A family of oligonucleotide tag sequences can be conjugated to a population of analytes most preferably polynucleotide sequences in several different ways including but not limited to direct chemical synthesis, chemical coupling, ligation, amplification, and the like. Sequence tags that have been synthesized with primer sequences can be used for enzymatic extension of the primer on the target for example in PCR amplification.

Detection of Single Nucleotide Polymorphisms Using Primer Extension

There are a number of areas of genetic analysis where families of non-cross-hybridizing sequences can be applied including disease diagnosis, single nucleotide polymorphism analysis, genotyping, expression analysis and the like. One such approach for genetic analysis, referred to as the primer extension method (also known as Genetic Bit Analysis (Nikiforov et al, Nucleic Acids Res.; 22, 4167-4175: 1994; Head et al Nucleic Acids Res.; 25, 5065-5071: 1997)), is an extremely accurate method for identification of the nucleotide located at a specific polymorphic site within genomic DNA. In standard primer extension reactions, a portion of genomic DNA containing a defined polymorphic site is amplified by PCR using primers that flank the polymorphic site. In order to identify which nucleotide is present at the polymorphic site, a third primer is synthesized such that the polymorphic position is located immediately 3′ to the primer. A primer extension reaction is set up containing the amplified DNA, the primer for extension, up to 4 dideoxynucleoside triphosphates (each labeled with a different fluorescent dye) and a DNA polymerase such as the Klenow subunit of DNA Polymerase 1. The use of dideoxy nucleotides ensures that a single base is added to the 3′ end of the primer, a site corresponding to the polymorphic site. In this way the identity of the nucleotide present at a specific polymorphic site can be determined by the identity of the fluorescent dye-labeled nucleotide that is incorporated in each reaction. One major drawback to this approach is its low throughput. Each primer extension reaction is carried out independently in a separate tube.

Universal sequences can be used to enhance the throughput of primer extension assay as follows. A region of genomic DNA containing multiple polymorphic sites is amplified by PCR. Alternatively, several genomic regions containing one or more polymorphic sites each are amplified together in a multiplexed PCR reaction. The primer extension reaction is carried out as described above except that the primers used are chimeric, each containing a unique universal tag at the 5′ end and the sequence for extension at the 3′ end. In this way, each gene-specific sequence would be associated with a specific universal sequence. The chimeric primers would be hybridized to the amplified DNA and primer extension is carried out as described above. This would result in a mixed pool of extended primers, each with a specific fluorescent dye characteristic of the incorporated nucleotide. Following the primer extension reaction, the mixed extension reactions are hybridized to an array containing probes that are reverse complements of the universal sequences on the primers. This would segregate the products of a number of primer extension reactions into discrete spots. The fluorescent dye present at each spot would then identify the nucleotide incorporated at each specific location. A number of additional methods for the detection of single nucleotide polymorphisms, including but not limited to, allele specific polymerase chain reaction (ASPCR), allele specific primer extension (ASPE) and oligonucleotide ligation assay (OLA) can be performed by someone skilled in the art in combination with the tag sequences described herein.

Kits Using Families Of Tag Sequences

The families of non cross-hybridizing sequences may be provided in kits for use in for example genetic analysis. Such kits include at least one set of non-cross-hybridizing sequences in solution or on a solid support. Preferably the sequences are attached to microparticles and are provided with buffers and reagents that are appropriate for the application. Reagents may include enzymes, nucleotides, fluorescent labels and the like that would be required for specific applications. Instructions for correct use of the kit for a given application will be provided.

EXAMPLES Example 4 Cross Talk Behavior of Sequence on Beads

A group of 100 sequences, randomly selected from Table II, was tested for feasibility for use as a family of minimally cross-hybridizing oligonucleotides. The 100 sequences selected are separately indicated in Table II along with the numbers assigned to the sequences in the tests.

The tests were conducted using the Luminex LabMAP™ platform available from Luminex Corporation, Austin, Tex., U.S.A. The one hundred sequences, used as probes, were synthesized as oligonucleotides by Integrated DNA Technologies (IDT, Coralville, Iowa, U.S.A.). Each probe included a C₆ aminolink group coupled to the 5′-end of the oligonucleotide through a C₁₂ ethylene glycol spacer. The C₆ aminolink molecule is a six carbon spacer containing an amine group that can be used for attaching the oligonucleotide to a solid support. One hundred oligonucleotide targets (probe complements), the sequence of each being the reverse complement of the 100 probe sequences, were also synthesized by IDT. Each target was labelled at its 5′-end with biotin. All oligonucleotides were purified using standard desalting procedures, and were reconstituted to a concentration of approximately 200 μM in sterile, distilled water for use. Oligonucleotide concentrations were determined spectrophotometrically using extinction coefficients provided by the supplier.

Each probe was coupled by its amino linking group to a carboxylated fluorescent microsphere of the LapMAP system according to the Luminex¹⁰⁰ protocol. The microsphere, or bead, for each probe sequence has unique, or spectrally distinct, light absorption characteristics which permits each probe to be distinguished from the other probes. Stock bead pellets were dispersed by sonication and then vortexing. For each bead population, five million microspheres (400 μL) were removed from the stock tube using barrier tips and added to a 1.5 mL Eppendorf tube (USA Scientific). The microspheres were then centrifuged, the supernatant was removed, and beads were resuspended in 25 μL of 0.2 M MES (2-(N-morpholino)ethane sulfonic acid) (Sigma), pH 4.5, followed by vortexing and sonication. One nmol of each probe (in a 25 μL volume) was added to its corresponding bead population. A volume of 2.5 μL of EDC cross-linker (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (Pierce), prepared immediately before use by adding 1.0 mL of sterile ddH₂₀ to 10 mg of EDC powder, was added to each microsphere population. Bead mixes were then incubated for 30 minutes at room temperature in the dark with periodic vortexing. A second 2.5 μL aliquot of freshly prepared. EDC solution was then added followed by an additional 30 minute incubation in the dark. Following the second EDC incubation, 1.0 mL of 0.02% Tween-20 (BioShop) was added to each bead mix and vortexed. The microspheres were centrifuged, the supernatant was removed, and the beads were resuspended in 1.0 mL of 0.1% sodium dodecyl sulfate (Sigma). The beads were centrifuged again and the supernatant removed. The coupled beads were resuspended in 100 μL of 0.1 M MES pH 4.5. Bead concentrations were then determined by diluting each preparation 100-fold in ddH₂O and enumerating using a Neubauer BrightLine Hemacytometer. Coupled beads were stored as individual populations at 8° C. protected from light.

The relative oligonucleotide probe density on each bead population was assessed by Terminal Deoxynucleotidyl Transferase (TdT) end-labelling with biotin-ddUTPs. TdT was used to label the 3′-ends of single-stranded DNA with a labeled ddNTP. Briefly, 180 μL of the pool of 100 bead populations (equivalent to about 4000 of each bead type) to be used for hybridizations was pipetted into an Eppendorf tube and centrifuged. The supernatant was removed, and the beads were washed in 1×TdT buffer. The beads were then incubated with a labelling reaction mixture, which consisted of 5×TdT buffer, 25 mM CoCl₂, and 1000 pmol of biotin-16-ddUTP (all reagents were purchased from Roche). The total reaction volume was brought up to 85.5 μL with sterile, distilled H₂O, and the samples were incubated in the dark for 1 hour at 37° C. A second aliquot of enzyme was added, followed by a second 1 hour incubation. Samples were run in duplicate, as was the negative control, which contained all components except the TdT. In order to remove unincorporated biotin-ddUTP, the beads were washed 3 times with 200 μL of hybridization buffer, and the beads were resuspended in 50 μL of hybridization buffer following the final wash. The biotin label was detected spectrophotometrically using SA-PE (streptavidin-phycoerythrin conjugate). The streptavidin binds to biotin and the phycoerythrin is spectrally distinct from the probe beads. The 10 mg/mL stock of SA-PE was diluted 100-fold in hybridization buffer, and 15 μL of the diluted SA-PE was added directly to each reaction and incubated for 15 minutes at 37° Celsius. The reactions were analyzed on the Luminex¹⁰⁰ LabMAP. Acquisition parameters were set to measure 100 events per bead using a sample volume of 50 μL.

The results obtained are shown in FIG. 6. As can be seen the Mean Fluorescent Intensity (MFI) of the beads varies from 840.3 to 3834.9, a range of 4.56-fold. Assuming that the labelling reactions are complete for all of the oligonucleotides, this illustrates the signal intensity that would be obtained for each type of bead at this concentration if the target (i.e., labelled complement) was bound to the probe sequence to the full extent possible.

The cross-hybridization of targets to probes was evaluated as follows. 100 oligonucleotide probes linked to 100 different bead populations, as described above, were combined to generate a master bead mix, enabling multiplexed reactions to be carried out. The pool of microsphere-immobilized probes was then hybridized individually with each biotinylated target. Thus, each target was examined individually for its specific hybridization with its complementary bead-immobilized sequence, as well as for its non-specific hybridization with the other 99 bead-immobilized universal sequences present in the reaction. For each hybridization reaction, 25 μL bead mix (containing about 2500 of each bead population in hybridization buffer) was added to each well of a 96-well Thermowell PCR plate and equilibrated at 37° C. Each target was diluted to a final concentration of 0.002 fmol/LL in hybridization buffer, and 25 μL (50 fmol) was added to each well, giving a final reaction volume of 50 μL. Hybridization buffer consisted of 0.2 M NaCl, 0.1 M Tris, 0.08% Triton X-100, pH 8.0 and hybridizations were performed at 37° C. for 30 minutes. Each target was analyzed in triplicate and six background samples (i.e. no target) were included in each plate. A SA-PE conjugate was used as a reporter, as described above. The 10 mg/mL stock of SA-PE was diluted 100-fold in hybridization buffer, and 15 μL of the diluted SA-PE was added directly to each reaction, without removal of unbound target, and incubated for 15 minutes at 37° C. Finally, an additional 35 μL of hybridization buffer was added to each well, resulting in a final volume of 100 μL per well prior to analysis on the Luminex¹⁰⁰ LabMAP. Acquisition parameters were set to measure 100 events per bead using a sample volume of 80 μL.

The percent hybridization was calculated for any event in which the NET MFI was at least 3 times the zero target background. In other words, a calculation was made for any sample where (MFI_(sample)−MFI_(zero target background))/MFI_(zero target background)≧3.

The net median fluorescent intensity (MFI_(sample)−MFI_(zero target background)) generated for all of the 10,000 possible target/probe combinations was calculated. As there are 100 probes and 100 targets, there are 100×100=10,0000 possible different interactions possible of which 100 are the result of perfect hybridizations. The remaining 9900 result from hybridization of a target with a mismatched probe. A cross-hybridization event is then defined as a non-specific event whose net median fluorescent intensity exceeds 3 times the zero target background. In other words, a cross-talk calculation is only be made for any sample where (MFI_(sample)−MFI_(zero target background))/MFI_(zero target background)≧3. Cross-hybridization events were quantified by expressing the value of the cross-hybridization signal as a percentage of the perfect match hybridization signal with the same probe.

The results obtained are illustrated in FIG. 7. The ability of each target to be specifically recognized by its matching probe is shown of the possible 9900 non-specific hybridization events that could have occurred when the 100 targets were each exposed to the pool of 100 probes, 6 events were observed. Of these 6 events, the highest non-specific event generated a signal equivalent to 5.3% of the signal observed for the perfectly matched pair (i.e. specific hybridization event).

Each of the 100 targets was thus examined individually for specific hybridization with its complement sequence as incorporated onto a microsphere, as well as for non-specific hybridization with the complements of the other 99 target sequences. Representative hybridization results for target (complement of probe 90, Table II) are shown in FIG. 8. Probe 90 was found to hybridize only to its perfectly-matched target. No cross-hybridization with any of the other 99 targets was observed.

The foregoing results demonstrate the possibility of incorporating the 1168 sequences of Table II, or any subset thereof, into a multiplexed system with the expectation that most if not all sequences can be distinguished from the others by hybridization. That is, it is possible to distinguish each target from the other targets by hybridization of the target with its precise complement and minimal hybridization with complements of the other targets.

Example 5 Tag Sequences Used in Sorting Polynucleotides

The family of non cross hybridizing sequence tags or a subset thereof can be attached to oligonucleotide probe sequences during synthesis and used to generate amplified probe sequences. In order to test the feasibility of PCR amplification with non cross hybridizing sequence tags and subsequently addressing each respective sequence to its appropriate location on two-dimensional or bead arrays, the following experiment was devised. A 24mer tag sequence can be connected in a 5′-3′ specific manner to a p53 exon specific sequence (20mer reverse primer). The connecting p53 sequence represents the inverse complement of the nucleotide gene sequence. To facilitate the subsequent generation of single stranded DNA post-amplification the tag-Reverse primer can be synthesized with a phosphate modification (PO₄) on the 5′-end. A second PCR primer can also be generated for each desired exon, represented by the Forward (5′-3′) amplification primer. In this instance the Forward primer can be labeled with a 5′-biotin modification to allow detection with Cy3-avidin or equivalent.

A practical example of the aforementioned description is as follows: For exon 1 of the human p53 tumor suppressor gene sequence the following tag-Reverse primer (SEQ ID NO: 1171) can be generated:                           222087                      222063 5′-P04-

-TCCAGGGAAGCGTGTCACCGTCGT-3′       Tag Sequence # 3                    Exon 1 Reverse

The numbering above the Exon-1 reverse primer represents the genomic nucleotide positions of the indicated bases. The corresponding Exon-1 Forward primer sequence (SEQ ID NO:1172) is as follows:           221873                      221896 5′-Biotin-TCATGGCGACTGTCCAGCTTTGTG-3′

In combination, these primers will amplify a product of 214 bp plus a 24 bp tag extension yielding a total size of 238 bp. Once amplified, the PCR product can be purified using a QIAquick PCR purification kit and the resulting DNA can be quantified. To generate single stranded DNA, the DNA is subjected to λ-exonuclease digestion thereby resulting in the exposure of a single stranded sequence (anti-tag) complementary to the tag-sequence covalently attached to the solid phase array. The resulting product is heated to 95° C. for 5 minutes and then directly applied to the array at a concentration of 10-50 nM. Following hybridization and concurrent sorting, the tag-Exon 1 sequences are visualized using Cy3-streptavidin. In addition to direct visualization of the biotinylated product, the product itself can now act as a substrate for further analysis of the amplified region, such as SNP detection and haplotype determination.

The Invader Assay is described in detail in U.S. Pat. Nos. 5,846,717 and 5,985,557. Briefly, the ability of the Invader technology to identify target nucleic acid sequences and in particular single base pair changes is dependent on the proper structure being formed, followed by subsequent recognition and cleavage of this structure by the Cleavase enzyme. For recognition by Cleavase III, the target sequence must be complementary to the primary probe, and there must be at least a 1 base “invasion” (overlap) of this structure by an upstream oligonucleotide. Cleavable “flaps’ can be created by invasion of an upstream oligonucleotide without primer extension, and the site of cleavage is determined by the extent to which the upstream oligonucleotide overlaps the 5′ region of the downstream oligonucleotide. Cleavage by the Cleavase enzyme is dependent on this invaded structure and is sensitive to single-base mismatches is positioned immediately upstream of the cleavage site. By adding overlapping pairs of oligonucleotide probes complementary to a predetermined region of target DNA, the cleavage of the downstream probes become a sensitive indicator of the presence of the target sequence. Further, reaction conditions have been established that allow multiple copies of the downstream oligonucleotide probe to be cleaved for each target sequence without temperature cycling, so as to amplify the cleavage signal and allow quantitative detection of target DNA at sub-attomole levels. Incorporation of the minimally cross-hybridizing sequences of the invention described herein into the probe that will be cleaved by the Cleavase enzyme allows detection of multiple target DNA sequences in a single experiment.

Definitions

Non-cross-hybridization: Describes the absence of hybridization between two sequences that are not perfect complements of each other.

Cross-hybridization: The hydrogen bonding of a single-stranded DNA sequence that is partially but not entirely complementary to a single-stranded substrate.

Homology or Similarity: How closely related two or more separate strands of DNA are to each other, based on their base sequences.

Analogue: The symbols A, G, T/U, C take on their usual meaning in the art here. In the case of T and U, a person skilled in the art would understand that these are equivalent to each other with respect to the inter-strand hydrogen-bond (Watson-Crick) binding properties at work in the context of this invention. The two bases are thus interchangeable and hence the designation of T/U. A chemical, which resembles a nucleotide base is an analogue thereof. A base that does not normally appear in DNA but can substitute for the ones, which do, despite minor differences in structure. Analogues particularly useful in this invention are of the naturally occurring bases can be inserted in their respective places where desired. Such an analogue is any non-natural base, such as peptide nucleic acids and the like that undergoes normal Watson-Crick pairing in the same way as the naturally occurring nucleotide base to which it corresponds.

Complement: The opposite or “mirror” image of a DNA sequence. A complementary DNA sequence has an “A” for every “T” and a “C” for every “G”. Two complementary strands of single stranded DNA, for example a tag sequence and its complement, will join to form a double-stranded molecule.

Complementary DNA (cDNA): DNA that is synthesized from a messenger RNA template; the single-stranded form is often used as a probe in physical mapping.

Oligonucleotide: Refers to a short nucleotide polymer whereby the nucleotides may be natural nucleotide bases or analogues thereof.

Tag: Refers to an oligonucleotide that can be used for specifically sorting analytes with at least one other oligonucleotide that when used together do not cross hybridize.

Similar Homology: In the context of this invention, pairs of sequences are compared with each other based on the amount of “homology” between the sequences. By way of example, two sequences are said to have a 50% “maximum homology” with each other if, when the two sequences are aligned side-by-side with each other so to obtain the (absolute) maximum number of identically paired bases, the number of identically paired bases is 50% of the total number of bases in one of the sequences. (If the sequences being compared are of different lengths, then it would be of the total number of bases in the shorter of the two sequences.) Examples of determining maximum homology are as follows:

Example 1

    *   * A-A-B-B-C-C     B-D-C-D-D-D (2 out of 4 paired bases are the same)       *   * A-A-B-B-C-C       B-D-C-D-D-D (2 out of 3 paired bases are the same)

In this case, the maximum number of identically paired bases is two and there are two possible alignments yielding this maximum number. The total number of possible pairings is six giving 33⅓% ( 2/6) homology. The maximum amount of homology between the two sequences is thus ⅓.

Example 2

* *     * A-A-B-B-C-A A-A-D-D-C-D (3 out of 6 paired bases are the same)

In this alignment, the number of identically paired bases is three and the total number of possibly paired bases is six, so the homology between the two sequences is 3/6(50%).           * A-A-B-B-C-A           A-A-D-D-C-D (1 out of 1 paired bases are the same)

In this alignment, the number of identically paired bases is 1, so the homology between the two sequences is ⅙ (16⅔%)

The maximum homology between these two sequences is thus 50%.

Block sequence: Refers to a symbolic representation of a sequence of blocks. In its most general form a block sequence is a representative sequence in which no particular value., mathematical variable, or other designation is assigned to each block of the sequence.

Incidence Matrix: As used herein is a well-defined term in the field of Discrete Mathematics. However, an incidence matrix cannot be defined without first defining a “graph”. In the method described herein a subset of general graphs called simple graphs is used. Members of this subcategory are further defined as follows.

A simple graph G is a pair (V, E) where V represents the set of vertices of the simple graph and E is a set of un-oriented edges of the simple graph. An edge is defined as a 2-component combination of members of the set of vertices. In other words, in a simple graph G there are some pairs of vertices that are connected by an edge. In our application a graph is based on nucleic acid sequences generated using sequence templates and vertices represent DNA sequences and edges represent a relative property of any pair of sequences.

The incidence matrix is a mathematical object that allows one to describe any given graph. For the subset of simple graphs used herein, the simple graph G=(V,E), and for a pre-selected and fixed ordering of vertices, V={v₁,v₂, . . . v_(n)}, elements of the incidence matrix A(G)=[a_(ij)] are defined by the following rules:

-   -   (1) a_(ij)=1 for any pair of vertices {v_(i),v_(j)} that is a         member of the set of edges; and     -   (2) a_(ij)=0 for any pair of vertices {v_(i),v_(j)}that is not a         member of the set of edges.         This is an exact unequivocal definition of the incidence matrix.         In effect, one selects the indices: 1, 2, . . . n of the         vertices and then forms an (n×n) square matrix with elements         a_(ij)=1 if the vertices v_(i) and v_(j) are connected by an         edge and a_(ij)=0 if the vertices v_(i) and v_(j) are not         connected by an edge.

To define the term “class property” as used herein, the term “complete simple graph” or “clique” must first be defined. The complete simple graph is required because all sequences that result from the method described herein should collectively share the relative property of any pair of sequences defining an edge of graph G, for example not violating the threshold rule that is, do not have a “maximum simple homology” greater than a predetermined amount, whatever pair of the sequences are chosen from the final set. It is possible that additional “local” rules, based on known or empirically determined behavior of particular nucleotides, or nucleotide sequences, are applied to sequence pairs in addition to the basic threshold rule.

In the language of a simple graph, G=(V, E), this means in the final graph there should be no pair of vertices (no sequence pair) not connected by an edge (because an edge means that the sequences represented by v_(i) and v_(j) do not violate the threshold rule).

Because the incidence matrix of any simple graph can be generated by the above definition of its elements, the consequence of defining a simple complete graph is that the corresponding incidence matrix for a simple complete graph will have all off-diagonal elements equal to 1 and all diagonal elements equal to 0. This is because if one aligns a sequence with itself, the threshold rule is of course violated, and all other sequences are connected by an edge.

For any simple graph, there might be a complete subgraph. First, the definition of a subgraph of a graph is as follows. The subgraph Gs=(Vs, Es) of a simple graph G=(V, E) is a simple graph that contains the subsets of vertices Vs of the set V of vertices and inclusion of the set Vs into the set V is immersion (a mathematical term). This means that one generates a subgraph Gs=(Vs,Es) of a simple graph G in two steps. First select some vertices Vs from G. Then select those edges Es from G that connect the chosen vertices and do not select edges that connect selected with non selected vertices.

We desire a subgraph of G that is a complete simple graph. By using this property of the complete simple graph generated from the simple graph G of all sequences generated by the template based algorithm, the pairwise property of any pair of the sequences (violating/non-violating the threshold rule) is converted into the property of all members of the set, termed “the class property”.

By selecting a subgraph of a simple graph G that is a complete simple graph, this assures that, up to the tests involving the local rules described herein, there are no pairs of sequences in the resulting set that violate the threshold rule, also described above, independent of which pair of sequences in the set are chosen. This feature is called the “desired class property”.

The present invention thus includes reducing the potential for non cross-hybridization behavior by taking into account local homologies of the sequences and appears to have greater rigor than known approaches. For example, the method described herein involves the sliding of one sequence relative to the other sequence in order to form a sequence alignment that would accommodate insertions or deletions. (Kane et al., Nucleic Acids Res.; 28, 4552-4557: 2000). TABLE I No. Assigned SEQ ID NO (1) Sequence in Example 3 1 GATTTGTATTGATTGAGATTAAAG 1 2 TGATTGTAGTATGTATTGATAAAG 2 3 GATTGTAAGATTTGATAAAGTGTA 3 4 GATTTGAAGATTATTGGTAATGTA 4 5 GATTGATTATTGTGATTTGAATTG 5 6 GATTTGATTGTAAAAGATTGTTGA 6 7 ATTGGTAAATTGGTAAATGAATTG 7 8 ATTGGATTTGATAAAGGTAAATGA 9 GTAAGTAATGAATGTAAAAGGATT 8 10 GATTGATTGATTGATTGATTTGAT 11 TGATGATTAAAGAAAGTGATTGAT 12 AAAGGATTTGATTGATAAAGTGAT 13 TGTAGATTTGTATGTATGTATGAT 10 14 GATTTGATAAAGAAAGGATTGATT 15 GATTAAAGTGATTGATGATTTGTA 11 16 AAAGAAAGAAAGAAAGAAAGTGTA 12 17 TGTAAAAGGATTGATTTGTATGTA 18 AAAGTGTAGATTGATTAAAGAAAG 19 AAAGTTGATTGATTGAAAAGGTAT 20 TTGATTGAGATTGATTTTGAGTAT 21 TGAATTGATGAATGAATGAAGTAT 15 22 GTAATGAAGTATGTATGTAAGTAA 23 TGATGATTTGAATGAAGATTGATT 16 24 TGATAAAGTGATAAAGGATTAAAG 17 25 TGATTTGAGTATTTGAGATTTTGA 18 26 TGTAGTAAGATTGATTAAAGGTAA 27 GTATAAAGGATTGATTTTGAAAAG 28 GTATTTGAGTAAGTAATTGATTGA 19 29 GTAAAAAGTTGAGTATTGAAAAAG 30 GATTTGATAAAGGATTTGTATTGA 31 GATTGTATTGAAGTATTGTAAAAG 20 32 TGATGATTTTGATGAAAAAGTTGA 33 TGATTTGAGATTAAAGAAAGGATT 21 34 TGATTGAATTGAGTAAAAAGGATT 22 35 AAAGTGTAAAAGGATTTGATGTAT 36 AAAGGTATTTGAGATTTGATTGAA 37 AAAGTTGAGATTTGAATGATTGAA 23 38 TGTATTGAAAAGGTATGATTTGAA 39 GTATTGTATTGAAAAGGTAATTGA 24 40 TTGAGTAATGATAAAGTGAAGATT 41 TGAAGATTTGAAGTAATTGAAAAG 25 42 TGAAAAAGTGTAGATTTTGAGTAA 26 43 TGTATGAATGAAGATTTGATTGTA 44 AAAGTTGAGTATTGATTTGAAAAG 27 45 GATTTGTAGATTTGTATTGAGATT 46 AAAGAAAGGATTTGTAGTAAGATT 29 47 GTAAAAAGAAAGGTATAAAGGTAA 30 48 GATTAAAGTTGATTGAAAAGTGAA 31 49 TGAAAAAGGTAATTGATGTATGAA 50 AAAGGATTAAAGTGAAGTAATTGA 33 51 ATGAATTGGTATGTATATGAATGA 34 52 TGAAATGAATGAATGATGAAATTG 35 53 ATTGATTGTGAATGAAATGAATTG 36 54 ATTGAAAGATGAAAAGATGAAAAG 37 55 ATTGTTGAAAAGTGTAATGATTGA 38 56 ATGATGTAATGAAAAGATTGTGTA 39 57 AAAGATTGAAAGATGATGTAATTG 58 ATTGATGAGTATATTGTGTAGTAA 41 59 AAAGATTGTGTAATTGATGATGAA 60 AAAGGTATATTGTGTAATGAGTAA 61 TGTAATGAGTATTGTAATTGAAAG 43 62 GTATAAAGAAAGATTGGTAAATGA 44 63 TTGAGTAATTGAATTGTGAAATGA 45 64 TGTATTGAATGAATTGTTGATGTA 46 65 TGTAATTGGTAAATGAGTAAAAAG 66 TGAATGAAATTGATGAGTATAAAG 67 GTAAGTAAATTGAAAGATTGATGA 49 68 GTAAATGATGATATTGGTATATTG 50 69 ATTGTTGATGATTGATTGAAATGA 51 70 ATTGTGAAGTATAAAGATGATTGA 52 71 ATGAAAAGTTGAGTAAATTGTGAT 72 ATGAATTGAAAGTGATTGAAAAAG 54 73 GTAAATTGATGAAAAGTTGATGAT 74 AAAGTGATGTATATGAGTAAATTG 56 75 GTAATGATAAAGATGATGATATTG 57 76 TTGAAAAGATTGGTAATGATATGA 77 AAAGTGAAAAAGATTGATTGATGA 59 78 ATTGATGAGATTGATTATTGTGTA 79 ATGAGATTATTGGATTTGTAGATT 60 80 TGAAGATTATGAATTGGTAAGATT 61 81 ATTGGATTATGAGATTATGATTGA 62 82 ATTGTTGAATTGGATTAAAGATGA 83 AAAGATGAGTAAGTAAATTGGATT 84 AAAGGTAAGATTATTGATGAAAAG 65 85 ATTGATGAGATTAAAGTTGAATTG 86 GATTATTGGATTATGAAAAGGATT 87 GATTTGTAATTGTTGAGTAAATGA 67 88 AAAGAAAGATTGTTGAGATTATGA 68 89 GTATAAAGGATTTTGAATTGATGA 90 TTGAGATTGTAAATGAATTGTTGA 91 GTATATTGATTGTGTAATGAAAAG 92 TGATATGAATTGGATTATTGGTAT 70 93 ATGAATGATGAATGATGATTATTG 94 ATGAATTGATTGGATTGTAATGAT 71 95 GATTGTAATTGAGTAAATTGATGA 96 GATTATTGGATTAAAGGTAAATGA 72 97 ATTGTTGAATTGATGAGATTTGAT 73 98 GATTATGAGTAAATTGATTGTGAT 99 GATTATTGTTGATGAATGATATTG 100 TGTAAAAGATTGAAAGGTATGATT 75 101 GTATTTAGATGAGTTTGTTAGATT 76 102 TGAAGTTATGTAATAGAAAGTGAT 103 GTATGTATTGTATGTAGTTAATTG 77 104 TGATATAGATAGTTAGATAGATAG 78 105 ATGATGATGTATTGTAGTTATGAA 79 106 TTAGTGAATGTATTAGTTGATGTA 107 GTTAGTTAGATTATTGTTAGTTAG 80 108 GTTAATTGTGTAGTTTGTTATTGA 109 GTTATGAAATAGTGATATTGTTAG 110 ATTGTTAGAAAGTGTAGATTAAAG 81 111 ATGAGTATGTTATTAGTGTATGTA 82 112 TGTAATAGTGAAGTTAGATTGTAT 83 113 ATTGATAGATGATTAGTTAGTTGA 84 114 ATGAGTTTGTTTATGAGATTAAAG 115 TGATGTTTGATTATGATGTAGTAT 85 116 ATGAGTTAGTTATGAATTAGATGA 117 ATTGTTAGTGATGTTAGTAATTAG 86 118 TGATGTAAGTATTGATGTTAGTTT 87 119 GATTGTAAATAGAAAGTGAAGTAA 88 120 ATTGTGTATGAAGTATTGTATGAT 121 ATAGTGATGTTATGAAGATTGTTA 122 TTAGATGAATTGTGAAGTATTTAG 90 123 GTAAGTTATGATTGATGTTATGAA 91 124 GTATTGATGTTTAAAGTGTAATAG 92 125 GATTGTAAGTAAGATTGTATATTG 126 GTTTGTATTTAGATGAATAGAAAG 93 127 GTTTGATTTGTAATAGTGATTGTA 128 TGTATGTAGTATTTAGAAAGATGA 129 ATGAATTGTGATAAAGAAAGTTAG 130 TTAGTGTAGTAAGTTTAAAGTGTA 95 131 GTATGATTGTTTGTAATTAGTGAT 132 GTTTAAAGTTAGTTGAGTTAGTAT 96 133 ATAGTGTATGTAGATTATGAGATT 97 134 TTGAATGATTAGTTGAGTATGATT 98 135 GTATGTAAGTTAGTATGATTTGAA 136 TGTAGTATATTGTTGAATTGTGAT 137 ATAGTGATTGTATGTATGATAAAG 138 TTAGTGATTGATGTATATTGAAAG 139 GTAAGATTATGAGTTATGATGTAA 140 GTTATGAAATTGTTAGTGTAGATT 99 141 GTTAGATTTGTAGTTTAAAGATAG 100 142 TTAGTGATTGAAATGATGTAGATT 143 AAAGTGTAGTTATTAGTTAGTTAG 144 AAAGAAAGTGTATGATGTTATTAG 145 GATTGTATATTGTGTATGATGATT 146 TTGAGATTGTTATGATATGAGTAT 147 ATGAGTATGATTGTTATGATGTTT 148 TGATTTAGTGAAATTGTGTATTAG 149 TGAATGTATGTAGTATGTTTGTTA 150 GTTAGTATTGATGATTATGAGTTA 151 GTATATTGTGATTTAGTTGAGATT 152 GTTAGTTTAAAGTTGAGATTGTTT 153 GTATATTGTTAGATGAGATTTGTA 154 TGATGTATGTTAGTTTATGAATGA 155 TGTAGTATGTAATGTAGTATTTGA 156 ATGAGTTATGTATTGAGTTAGTAT 157 TGTATGATGATTATAGTTGAGTAA 158 ATTGATGAATGAGTTTGTATAAAG 159 TTGAGTTTATGATTAGAAAGAAAG 160 TGATATTGATGAGTTAGTATTGAA 161 ATAGAAAGTGAAATGAGTATGTTA 162 TTGATGTAGATTTGATGTATATAG 163 TTGAGATTATAGTGTAGTTTATAG 164 TGATGTTAGATTGTTTGATTATTG 165 TGTATTAGATAGTGATTTGAATGA 166 GATTATGATGAATGTAGTATGTAA 167 TGAATGATTGATATGAATAGTGTA 168 GTAATGATTTAGTGTATTGAGTTT 169 TGTAGTAATGATTTGATGATAAAG 170 TGAAGATTGTTATTAGTGATATTG 171 GTATTTGAATGATGTAATAGTGTA 172 GTATATGATGTATTAGATTGAAAG 173 AAAGTTAGATTGAAAGTGATAAAG 174 GTAAGATGTTGATATAGAAGATTA 9 175 TAATATGAGATGAAAGTGAATTAG 176 TTAGTGAAGAAGTATAGTTTATTG 13 177 GTAGTTGAGAAGATAGTAATTAAT 178 ATGAGATGATATTTGAGAAGTAAT 179 GATGTGAAGAAGATGAATATATAT 180 AAAGTATAGTAAGATGTATAGTAG 14 181 GAAGTAATATGAGTAGTTGAATAT 182 TTGATAATGTTTGTTTGTTTGTAG 28 183 TGAAGAAGAAAGTATAATGATGAA 184 GTAGATTAGTTTGAAGTGAATAAT 32 185 TATAGTAGTGAAGATGATATATGA 186 TATAATGAGTTGTTAGATATGTTG 187 GTTGTGAAATTAGATGTGAAATAT 188 TAATGTTGTGAATAATGTAGAAAG 40 189 GTTTATAGTGAAATATGAAGATAG 42 190 ATTATGAAGTAAGTTAATGAGAAG 47 191 GATGAAAGTAATGTTTATTGTGAA 192 ATTATTGAGATGTGAAGTTTGTTT 48 193 TGTAGAAGATGAGATGTATAATTA 53 194 TAATTTGAGTTGTGTATATAGTAG 195 TGATATTAGTAAGAAGTTGAATAG 196 GTTAGTTATTGAGAAGTGTATATA 55 197 GTAGTAATGTTAATGAATTAGTAG 58 198 GTTTGTTTGATGTGATTGAATAAT 199 GTAAGTAGTAATTTGAATATGTAG 64 200 GTTTGAAGATATGTTTGAAGTATA 201 ATGATAATTGAAGATGTAATGTTG 202 GTAGATAGTATAGTTGTAATGTTA 66 203 GATGTGAATGTAATATGTTTATAG 69 204 TGAAATTAGTTTGTAAGATGTGTA 74 205 TGTAGTATAAAGTATATGAAGTAG 63 206 ATATGTTGTTGAGTTGATAGTATA 89 207 ATTATTGAGTAGAAAGATAGAAAG 94 208 GTTGTTGAATATTGAATATAGTTG 209 ATGAGAAGTTAGTAATGTAAATAG 210 TGAAATGAGAAGATTAATGAGTTT

TABLE II No. in Sequence SEQ ID NO: Ex 4 AAATTGTGAAAGATTGTTTGTGTA 1 1 GTTAGAGTTAATTGTATTTGATGA 2 — ATGTTAAAGTAAGTGTTGAAATGT 3 — TGATGTTAGAAGTATATTGTGAAT 4 — TTTGTGTAGAATATGTGTTGTTAA 5 — ATAAGTGTAAGTGAAATAAGAAGA 6 — AAGAGTATTTGTTGTGAGTTAAAT 7 — GTGTTTATGTTATATGTGAAGTTT 8 — AAAGAGAATAGAATATGTGTAAGT 9 — TATGAAAGAGTGAGATAATGTTTA 10 — ATGAGAAATATGTTAGAATGTGAT 11 — TTAGTTGTTGATGTTTAGTAGTTT 12 — GTAAAGAGTATAAGTTTGATGATA 13 — AAAGTAAGAATGATGTAATAAGTG 14 — GTAGAAATAGTTTATTGATGATTG 15 — TGTAAGTGAAATAGTGAGTTATTT 16 2 AAATAGATGATATAAGTGAGAATG 17 — ATAAGTTATAAGTGTTATGTGAGT 18 — TATAGATAAAGAGATGATTTGTTG 19 — AGAGTTGAGAATGTATAGTATTAT 20 — AAGTAGTTTGTAAGAATGATTGTA 21 — TTATGAAATTGAGTGAAGATTGAT 22 — GTATATGTAAATTGTTATGTTGAG 23 — GAATTGTATAAAGTATTAGATGTG 24 4 TAGATGAGATTAAGTGTTATTTGA 25 — GTTAAGTTTGTTTATGTATAGAAG 26 — GAGTATTAGTAAAGTGATATGATA 27 — GTGAATGATTTAGTAAATGATTGA 28 — GATTGAAGTTATAGAAATGATTAG 29 — AGTGATAAATGTTAGTTGAATTGT 30 — TATATAGTAAATGTTTGTGTGTTG 31 — TTAAGTGTTAGTTATTTGTTGTAG 32 — GTAGTAATATGAAGTGAGAATATA 33 — TAGTGTATAGAATGTAGATTTAGT 34 — TTGTAGATTAGATGTGTTTGTAAA 35 — TAGTATAGAGTAGAGATGATATTT 36 — ATTGTGAAAGAAAGAGAAGAAATT 37 7 TGTGAGAATTAAGATTAAGAATGT 38 — ATATTAGTTAAGAAAGAAGAGTTG 39 — TTGTAGTTGAGAAATATGTAGTTT 40 — TAGAGTTGTTAAAGAGTGTAAATA 41 — GTTATGATGTGTATAAGTAATATG 42 — TTTGTTAGAATGAGAAGATTTATG 43 10 AGTATAGTTTAAAGAAGTAGTAGA 44 — GTGAGATATAGATTTAGAAAGTAA 45 — TTGTTTATAGTGAAGTGAATAGTA 46 — AAGTAAGTAGTAATAGTGTGTTAA 47 — ATTTGTGAGTTATGAAAGATAAGA 48 — GAAAGTAGAGAATAAAGATAAGAA 49 — ATTTAAGATTGTTAAGAGTAGAAG 50 — GTTTAAAGATTGTAAGAATGTGTA 51 — TTTGTGAAGATGAAGTATTTGTAT 52 — TGTGTTTAGAATTTAGTATGTGTA 53 — GATAATGATTATAGAAAGTGTTTG 54 — GTTATTTGTAAGTTAAGATAGTAG 55 — AGTTTATTGAAAGAGTTTGAATAG 56 — TTGTGTTTATTGTGTAGTTTAAAG 57 — ATTGTGAGAAGATATGAAAGTTAT 58 — TGAGAATGTAAAGAATGTTTATTG 59 13 ATGTGAAAGTTATGATGTTAATTG 60 — GTTTAGTATTAGTTGTTAAGATTG 61 — GATTGATATTTGAATGTTTGTTTG 62 14 TGAATTGAAAGTGTAATGTTGTAT 63 — GATTGTATTGTTGAGAATAGAATA 64 — AAATTTGAGATTTGTGATAGAGTA 65 — GTAATTAGATTTGTTTGTTGTTGT 66 — GTTTGTATTGTTAGTGAATATAGT 67 — ATGTAGTAGTAGATGTTTATGAAT 68 — TGTTTAAAGATGATTGAAGAAATG 69 — TGTGATAATGATGTTATTTGTGTA 70 — ATAGTTGTGAGAATTTGTAATTAG 71 — ATAGATGTAAGAGAAATTGTGAAA 72 — AGATTAAGAGAAGTTAATAGAGTA 73 — GAAGTAAATTGTGAATGAAAGAAA 74 — AATGTAAGAAAGAAGATTGTTGTA 75 — TTTGATTTATGTGTTATGTTGAGT 76 — GTATTGAGAAATTTGAAGAATGAA 77 — GAATTGTATGAAATGAATTGTAAG 78 — TATTGTAGAAGTAAAGTTAGAAGT 79 — TTTATGTAATGATAAGTGTAGTTG 80 — ATATAGTTGAAATTGTGATAGTGT 81 — ATAAGAAATTAGAGAGTTGTAAAG 82 — GAATTGTGAAATGTGATTGATATA 83 — AAATAAGTAGTTTAATGAGAGAAG 84 — GATTAAAGAAGTAAGTGAATGTTT 85 — TATGTGTGTTGTTTAGTGTTATTA 86 — GAGTTATATGTAGTTAGAGTTATA 87 — GAAAGAAAGAAGTGTTAAGTTAAA 88 — TAGTATTAGTAAGTATGTGATTGT 89 — TTGTGTGATTGAATATTGTGAAAT 90 — ATGTGAAAGAGTTAAGTGATTAAA 91 — GATTGAATGATTGAGATATGTAAA 92 — AAGATGATAGTTAAGTGTAAGTTA 93 17 TAGTTGTTATTGAGAATTTAGAAG 94 — TTTATAGTGAATTATGAGTGAAAG 95 — GATAGATTTAGAATGAATTAAGTG 96 18 TTTGAAGAAGAGATTTGAAATTGA 97 — ATGAATAAGAGTTGATAAATGTGA 98 — TGTTTATGTAGTGTAGATTGAATT 99 — TTTAAGTGAGTTATAGAAGTAGTA 100 19 GATTTATGTGTTTGAAGTTAAGAT 101 — TAGTTAGAGAAAGTGATAAAGTTA 102 — GTAATGATAATGAAGTGTATATAG 103 — AATGAAGTGTTAGTATAGATAGTA 104 — TAAATTGAGTTTGTTTGATTGTAG 105 — TAATGAAGAATAAGTATGAGTGTT 106 — AAATGTAATAGTGTTGTTAGTTAG 107 — AGAGTTAGTGAAATGTTGTTAAAT 108 — GAAATAGAAATGTATTGTTTGTGA 109 — AGTTATAAGTTTGTGAGAATTAAG 110 — GAGTTTATAGTTAGAATATGTTGT 111 — AGAGTTATTAGAAGAAGATTTAAG 112 — GAGTTAATGAAATAAGTATTTGTG 113 — ATGATGAATAGTTGAAGTATATAG 114 — ATAGATATGAGATGAAAGTTAGTA 115 — TATGTAAAGAAAGTGAAAGAAGAA 116 — TGAATGTAGAAATGAATGTTGAAA 117 — AATTGAATAGTGTGTGAGTTTAAT 118 — AGATATTGTTTGATTAATGAAGAG 119 — AAAGTTGTAAAGTTGAAGATAAAG 120 — GTTAAGAGATTATGAGATGTATTA 121 — AGAAGATATAAGAAGATTGAATTG 122 — GTAGAAATTTGAATTGATGTGAAA 123 — AAGAGTAGATTGATAAGTATATGA 124 — TGATATAGTAGTGAAGAAATAAGT 125 22 AGATAATGATGAGAAATGAAGATA 126 — ATGTGAAAGTATTTGTGATATAGT 127 — AATAAGAGAATTGATATGAAGATG 128 23 TAAGTGTATTTAGTAGAATGAAGT 129 — TATGTTAGATTTGTTGAGATTGAT 130 — AGTTTGTATGAAGAGATAGTATTT 131 — GAGAAATGTTATGTATTTAGTAGT 132 — TATGTGAGAATGTGTTTGATTTAA 133 — GTATGTTTGTTTATAGAATGTATG 134 — GAGTATATAGAAGAAAGAAATTTG 135 — ATGAGTGAAGTAAATGTAGTTATT 136 — TTAAGAAGTGAGTTATTGTGATAT 137 — ATGAAATGAGAATATTGTTGTTTG 138 — GATTAATGATTATGTGAATTGATG 139 — GAAATGTTAAAGATATGAAAGTAG 140 — TATTGTTGATTTGATATTAGTGTG 141 — TTTATGTTTGTGTATGTAAGTAGT 142 — AATTGAAAGAATTGTGTGAATTGA 143 — TGAGTTTGAATTTGTTTGAGTAAT 144 — GATGTATAATGATGTGTGTAAATT 145 — ATGTGAGAGAAGAATTTGTTTATT 146 — GTGATAAAGTATTGTTGATAGAAA 147 — GAAGTAGAATAGAAAGTTAATAGA 148 — TTGTGTAGTTAAGAGTTGTTTAAT 149 24 TAGTAGTAAGTTGTTAGAATAGTT 150 — AATTTGAAGTATAATGAATGTGTG 151 — TAGAAATTGTAGTATTTGAGAGAA 152 — TGTATATGTTAATGAGATGTTGTA 153 25 TATTTGATAAGAGAATGAAGAAGT 154 26 TTGAATAGTGTAATGAATATGATG 155 — GTAGTTTGTGAATAGAATTAGTTT 156 — AAAGATGATTGTAATTTGTGTGAA 157 — GAAGATTGTTGAGTTAATAGATAA 158 — AGATTATGTAGTGATGTAAATGTT 159 — GAATTTAGATGTAGATATGAATGT 160 — GATAGAAGTGTATTAAGTAAGTTA 161 — TATGAATTATGAGAAGAATAGAGT 162 — TTTGTTATGAAGTGATTTGTTTGT 163 — GTAAAGATTGTGTTATATGAAATG 164 — TTGTGATAGTAGTTAGATATTTGT 165 28 GAATTAAGATAAAGAAGAGAAGTA 166 — GATTGTAGAATGAATTTGTAGTAT 167 — AAATAAGAGAGAGAATGATTTAGT 168 — AATTATGTGAATAGATTGTTGAAG 169 — TTAAGATTTATGTGATAGTAGAGT 170 — TTAAAGATAGTGTTTGTTGTGTTA 171 — TATTGATTTATGAAGAGTATAGTG 172 — AAATTTGATGAGTAGTTTAAGAGA 173 — ATAAAGTTGTTTGATGTTTGAATG 174 — GATTGTGATGAATAATGTTATTGA 175 — GATGAAGAAATATGATATGAATAG 176 — TTAAAGTTATTGAAGTGAAGTTGA 177 — TTGTAAGAAATAGAGATTTGTGTT 178 — GAGATTGAGTTTAAGTATTAGATT 179 — AGTGATAATAGAATGATAAATGTG 180 — GATAATAGTGAATTTGAGTTGTAT 181 — AGATATTTGTAGTAGAAAGTATGT 182 — GTTATGAATGTTGAATTTGAATGT 183 — ATGAAAGATTTAGTTGTGAGATAT 184 30 AAATAGAGAAGTTATGATGTGATA 185 — TTAGTGAGAAATGTTTAATGTGAT 186 — TGAAGAATATGTGAAATTAGTTTG 187 — GTTTGATAGTTTAATGAGTATTGA 188 — GTTGTAAGTAATGATAAAGTATGA 189 — TAAGAGTAGTAATTGTTGTTTAGA 190 — TTTGAGAGAGTATGTATGATTATT 191 — ATTGATTGTGAATTAGATAGAAGA 192 — GATTAGTATTTAGTAGTAATAGAG 193 31 TATGTATTAGAGATATTGAAAGTG 194 — TATGTGAAAGTAATGATAAATGAG 195 — GTAATTAGTAATGATTTGAATGAG 196 — GTTTATTGTAAAGATGTAAGTGAA 197 — TAGTAGAATTGTTGTTAAAGAATG 198 32 TATTGTTAGTTATGTAGTGTGTAA 199 — GAGTGAAAGTTATATGAAAGTATA 200 — ATATAGAAGTTGATGAGTTTATGA 201 — TTTAGAAGTAAGAATAAGTGAGTA 202 — TGTGTATAAGATATTTGTAAGAAG 203 — TAGAAGAGTTGTATTGTTATAAGT 204 — GTGTTATTAGTTTAAGTTAGAGTA 205 — AATATAGTGATGTGAAATTGAATG 206 — TTAGAGAATAGAGTGATTATAGTT 207 — GAAGTGAGTTAATGATTTGTAAAT 208 — AATGTAAAGTAAAGAAAGTGATGA 209 33 GTTAGTTATGATGAATATTGTGTA 210 34 AAATGAGTTAGAGTAGAATTATGT 211 — GATATAGAAGATTAGTTAGTGATA 212 — ATAGTTTGTTGAGATTTATGAGTA 213 — TAGAATAGTTAGTAGTAAGAGTAT 214 — GAATTTGTATTGTGAAGTTTAGTA 215 — GTAGTAAGAAGAGAATTAGATTAA 216 — AATGTGTTATGTATGTAAATAGTG 217 — GAATTAGTTAGAGTAAATTGTTTG 218 — GAAATTGAAGATAGTAAGAAATGA 219 — GTGTATTATGTGATTTATGATAGA 220 — TATTATGAGAAAGTTGAATAGTAG 221 35 TATGTATTGTATTGAGTAGATGAA 222 — GTGATTGAATAGTAGATTGTTTAA 223 36 AGTAAGTTGTTTGATTGAAATTTG 224 — GAAGTTTGATTTAAGTTTAAGAAG 225 — GAGAAGATAAATGATATTGTTATG 226 — ATGATGAGTTGTTAATAGTTAGTT 227 — TATGATATTTGAAGAGTGTTAAGA 228 — GAGATGATTAAAGTGATTTATGAA 229 — ATAGTTAAGAGTGATGAGAATAAA 230 — TTTATTGTTAGATAAAGAGTTGAG 231 — AGAATATTGATAGTTGAAGTTGAA 232 — TAGTGTAAAGTGTAGATTGTAAAT 233 — AGTAGTGATATGATTTGAATATTG 234 — TGTATTGAATTAGAATAGTGAGAA 235 — TGATATGAGATAGAAGTTTAATGT 236 — GAAGAAGTAAGTATAAAGTAAATG 237 — TTTAAGTGTGATAAGAAAGATAGA 238 — TATTGTTGAATGTGTTTAAAGAGA 239 38 GAATAATGATGAGATGATTATTGA 240 — TAGAGAAAGAGAGAATTGTATTAA 241 39 ATGTATAATGAGATATGTTTGTGA 242 — AATAGATAAGATTGATTGTGTTTG 243 40 TTTGATGATAATAGAAGAGAATGA 244 — AGATGAATAAGTTGTGAATGTTTA 245 — AGATGAAAGAAAGTGTAGAATATT 246 — TGTTAAATGTATGTAGTAATTGAG 247 41 TAGTAGTGTGAAGTTATTTGTTAT 248 — AGTGAATGTTTGTAAAGAGTTTAA 249 — GATAAATGAGAATTGAGTAATTGT 250 — TGATGAGAAATTGTTTAAGTGTTT 251 — AAATAAGTAGTGTGAGTAATAGTA 252 — TATGAAATATGTGATAGTAAGAGA 253 — ATTGTAAGAGTGATTATAGATGAT 254 — AGAGTAAGAATGAAAGAGATAATA 255 — TAAGTAAGTAGATGTTAAAGAGAT 256 — AAATAGAAAGAATTGTAGAGTAGT 257 — ATAGATTTAAGTGAAGAGAGTTAT 258 42 GAATGTTTGTAAATGTATAGATAG 259 43 AAATAGAATGAGTAGTGAAATATG 260 — TTGAATTATGTAGAGAAAGTAAAG 261 — TAGTAAATTGAGAGTAGTTGAATT 262 — TGTAAAGTGTTTATAGTGTGTAAT 263 — ATATGATTTGAGATGAGAATGTAA 264 — AATATTGATATGTGTTGTGAAGTA 265 — AGTGAGATTATGAGTATTGATTTA 266 44 TTGTATTTAGATAGTGAGATTATG 267 — ATAGAAATGAAAGATAGATAGAAG 268 — GATTGTATATGTAAAGTAGTTTAG 269 — TATGAATGTTATTGTGTGTTGATT 270 45 GATATTAGTAGAGTAAGTATATTG 271 — TGAGATGAATTTGTGTTATGATAT 272 — TATGAATGAAGTAAAGAGATGTAA 273 — GAGTGAATTTGTTGTAATTTGTTT 274 — AGAAATTGTAGAGTTAATTGTGTA 275 — GTGTTAATGAAAGTTGTGAATAAT 276 — TGTGATTTGTTAAGAAGATTAATG 277 — AGTAGTATTGTAAAGTATAAAGAG 278 — TGATTGTTGTATAGTTATTGTGTA 279 — GATTGTAGTTTAATGTTAAGAATG 280 — ATGAAATAAGAAATTGAGTAGAGA 281 — TATGATGATATTTGTTGTATGTGT 282 — TTTAGAGTTTGATTAGTATGTTTG 283 — AATAAGAGATTGTGATGAGAAATA 284 — AATGAATAGAATAGAGAATGTAGA 285 — GTAGTAGTAATTTGAATGTTTGAA 286 47 AGTGAGTAATTGATTGATTGTTAA 287 — GAATAATGTTTAGTGTGTTTGAAA 288 — ATATGAAAGTAGAGAAAGTGTTAT 289 — TGAGTTATTGTATTTAGTTTGAAG 290 — TAGTTGAGTTTAAAGTTGAAAGAA 291 — TAAAGAGTGATGTAAATAGAAGTT 292 — TGTAGTGTTTAGAGTAAGTTATTA 293 — AGAGATTAATGTGTTGAAAGATTA 294 — GTAATAAGTTGTGAAAGAAGATTA 295 — GAGATGTTATAGATAATGAAAGAA 296 — TTTAGTTGATTGTTGAATAGAGTA 297 — ATTATTGAAAGTAGATGTTAGATG 298 — TTTATGTGTGATTGAGTGTTTAAT 299 — TATTTAGTTAGATAGATAGAGAGT 300 — ATGTGTTTATGTGAAAGATTTGTA 301 — ATAGTAATTAGAAGAGAAGAATGT 302 — TATGAGTGATTAGAATTGTATTTG 303 — TTAATGTATTGTTTAAAGAGTGTG 304 — ATAGAGAATTAAGAATTGTTTGAG 305 — GTTATAAGTAGAAATGTATAGAAG 306 — AGTAATTAGTTTGAAATGTGTAGT 307 — GAAAGATTATGATTGTAAAGTGAT 308 — GTAAGATTAGAAGTTAATGAAGAA 309 48 GAGAATGTTGAATAAGAAGTAATT 310 — TTAAGAGTGTTTGAATAGTGTTTA 311 — ATAAAGAAAGAGTATGAGATTATG 312 — AGTTATTGATTGAAGATGAGAAAT 313 — GTTTGTGTTTGTATAAGTTGTTAA 314 50 TTGTATGTGAGTTTAGATTAATGA 315 — TAGTTAAAGTATAGTTGTTTGAGT 316 — AAATTTGTGTTGAGATTTGTATAG 317 — TATTAGTGTTATGATAAAGAGAAG 318 — TATAAGAAGTAATTTGAGAAGAGT 319 — TAAGTTGAGATGTTTGTTTGATAA 320 — GTGTAGATTTATGAATTGAGTAAT 321 — TATAGAGAAGTGTTTAGTTGTATA 322 — ATAAAGAAGAATAGTTGTTGTGTA 323 — AGATTGAAATAGATTAGAAAGTTG 324 — GTTGTTATAAGAAATAGTTTGTTG 325 — AGAAATAGAGTAAGAGTGTTTAAA 326 — AGAGATAGTAGTAAATAGTTATTG 327 — AAATGATTGTGTAAGTTATGTATG 328 — AAGAAGTAAGAGAGAAATTTGAAT 329 — GTGTGTATTTAGTTGATAATTGAT 330 — ATTGTTGTTGTTGAGAAATGTATT 331 — AGATAAGTTAAAGTAAAGAGAATG 332 — TAGTTGAAGTTAGTTTAAGTGTTA 333 — AGTAAGAATGTAATATGATGATAG 334 — ATGAGATTGAAAGATTTATGAATG 335 — TGATTGAATTAGAGAGAATGTATA 336 — AGTTAGTAAGAGAATATAGTGAAT 337 — ATTAAGATTGTATAGTTAGTGATG 338 — GAGATAAAGAATTGAAATAGAAGA 339 — AGAGTAAATGTTAAGAAAGAAGTT 340 — AAAGTTTGTTATGTGTGAAGAATT 341 — ATTGTGTTTAAGAAATATGATGAG 342 — TATTGAAATGAGATGTATGTAGTT 343 — ATTTGTGTGATGTTTGAAATATGA 344 — TAAGATAATAGTGAGAGAAATTGA 345 — ATTTATGATTAGTGTAAGTGTTGT 346 — GATTAAGAATAAAGTGTGAAGAAT 347 — GTAATTGATGAAGAGTTAGTTTAT 348 — TGTGTTATGTTATAAGAAGTGATA 349 — AGAGAAATTGAATTTAGAAATGTG 350 — TTATTGAATGTGAGAAAGTATTTG 351 — TGTTAATGAGAAGATAATGATAGT 352 — GAAAGTATTTGTTGATTATTGTTG 353 — TAGTTTATGTAGTTAATTGTTGAG 354 — GTTGAAAGATAGTTTGATATGTAT 355 — TTAGAAGATAGATTATTGAGAAAG 356 — AATAATGTTGTGAAATAGATGTGA 357 56 AGTAAGAAAGTTTAGTTTAGTTAG 358 — TAGTTTAATGAGATGTTTGATATG 359 — TTAAAGATGTTAAAGAATGAGTGA 360 — AAAGTGTGTATATGTTAGAAAGTA 361 — ATTAAGTTATGTGTTTATGTGTTG 362 — TTTGAAGAAGTGTTTGTATTATGT 363 — TGTTAAGAAGTTTAGTTAAAGTTG 364 — TTTAAGTATAAGATTGTGTGAGAT 365 — AGATATTTGATAGATAGAAGAAAG 366 — ATTTAGAGTTGTAAGAAGATATTG 367 — GAGAAATTGTAATTGTTAGAGTAT 368 — GAAGTATATGTTAAGATGTAATAG 369 — AATATTGAAGATGTAGTGAGTTAT 370 — GAGTTTAGAAATGATAAAGAATTG 371 — TAAGAAATGAGTTATATGTTGAGA 372 60 TTGATATAAGAAGTTGTGATAAGT 373 — AAGTGTTTAATGTAAGAGAATGAA 374 61 GTTGTGAGAATTAGAAATAGTATA 375 — TTTAGTTTGATGTGTTTATGAGAT 376 — GTAATTGAAAGTATGAGTAGTAAT 377 — TAGTTGAATAAGATTGAGAGAAAT 378 — TTAAGTGAAGTGTTGTTTATTGAA 379 — ATTGATTTGTTGAAATAAGTGTTG 380 — TGAATTGTTGATAAGTTATGAAGA 381 — GTTTGTTATTGAGTAAGTTGAATT 382 — TGATTTAGTATGTATTAGAGTTGA 383 — TAAATAGAGATGAGAATAAGAAAG 384 — AGAATGTTATATGTAGAGAAATTG 385 — ATTTATGTAGTTGAGAGTGATAAA 386 — GTAAAGATAGTTTGAGTAATTTGA 387 — GAAATAGTATAATGTTAAGTGAGA 388 — ATTGTATATTGTGTTGAAGAAAGT 389 — GAGTTAAGTGTAAATGAAATGTAA 390 — ATAGATTGTGTGAAAGAAAGAATT 391 — TTAATAGAAGTTTGTAGTATGATG 392 — TTGTATGTGAGAATAAAGTTTAGT 393 — GTGATTAGATATGATGATATGAAT 394 — TGAAGAAGAATTTAGATTTGTAAG 395 — TGTATGATTATTGATTAGTGTGTT 396 — TGTGAAAGAGAATGATAGATATTT 397 — AATTGAAATGAGTGTGTTTAAGAA 398 — ATTATAGAGTTAGTTTAGAATGAG 399 — AAAGATAGAAATTGAGTGTATGAT 400 — GTAGTTTGTTAATGTTGTATAATG 401 — AGAGATATTAGAATGTAAGAATAG 402 64 AGAAGTTTGAAATATGATAGAATG 403 — TAGAATGTAAAGTTTAGTATAGAG 404 — AGTAGATGTATGTTAATGTGAATA 405 — TGAAAGTGAAATATGAAATGTTGT 406 — ATAGTATATTGAGTTTGTATGAAG 407 — GAAGAAATGTTTGTAGAATAAGTA 408 — AATGAGTATTGAAGAAATGTATAG 409 — GTGATAGAATTTGTGTTTAATGAA 410 66 TGTAGTATGAAGAATAATGAAATG 411 — ATAGAAGTTAATGATAATTGTGTG 412 — GTGATTGTAAGTAAGTAAAGATAA 413 — TATGTAGTTTGTGTTATTTGAAGA 414 — TGAGTAAGTTTGTATGTTTAAGTA 415 67 TAAATGTATGAGTGTGTAAAGAAA 416 — GTAAGAGTATTGAAATTAGTAAGA 417 68 GTTGAGTGTAAAGATTATTGATAA 418 — AGTATGAGTTATTAGATAAAGTGA 419 — ATTTGTTATAGAGTTGTGTTGTAT 420 — TAATTAGTAGTGTGTTGAAATTTG 421 — TGTATTGAGATTGTTATTGTATTG 422 — GTTATTAGAAGAGATAATTGAGTT 423 — TTGAGTTGTGATTAAGTAGTATAT 424 — GATAGTATAATGATTGAAGTAATG 425 — GTGAAAGATATTTGAGAGATAAAT 426 — AGTTATGATTTGAAGAAATTGTTG 427 — GTAAGTATTTGAATTTGATGAGTT 428 — TAATAGTGTTATAAGTGAAAGAGT 429 — AAATGAATTGATGTGTATATGAAG 430 — AGAAAGTGAGTTGTTAAGTATTTA 431 — TTTATGTGTGAATTGTGTATATAG 432 — GTAATATGATAGAAATGTAAAGAG 433 — GAGAATTGTTTAAAGATAGTTGTA 434 — GAATTTGTTAAGAATGAGTTTGAT 435 — ATAGTGATGATTAAAGAGAATTTG 436 — ATAGATGTTTAGTTGAGATTATTG 437 — AAGAGTGTAAATAGAAAGTGATAT 438 — TGTGTATTGATTGTTGAGATAAAT 439 — TAGTATAGTGAGAAAGAGTTAAAT 440 — AAAGATAAGAAAGAGATGATGTTT 441 — GAAGTTATTGAAATAGAGAAGTAT 442 — ATGTATGTATAGAAAGAGTAAATG 443 — GATGTTTGTAAAGATTGAAATTGA 444 — AATTTAGAGAGTATTTGTGTTGTA 445 — AATTTGTTTGAAAGAAAGTAAGTG 446 — AAAGAGTAGTGTTATTGTTAGATA 447 — GTATGTTGTATATGTTGTTGATAT 448 — GTAGAATTTGTTGAGTATTTGTAA 449 — ATGAATTTAGTTAGTGTAAGAAAG 450 — ATGATAAGAAATGTTGATGAAGTA 451 — TTGATGATGAAGATAATGTAGATA 452 — AGATGATATGATATAGATTAGATG 453 — TTGAAAGTTAGAAAGATAGATGTT 454 — GTTTAATGTTAGTTAGAAAGTAAG 455 — GAGATTTAAGTTTGAAGTGAAATA 456 — TTTGTTAGTAGTTGTTATAAGAGA 457 — TATGAGAATAGTTTGTTAGTGAAT 458 — TTGAAAGTTTAAAGAAGAGATAAG 459 — AAGTGAGTTGAAATGAAATATGTT 460 — GTTAGAAATGAAATGAGTAGTTAT 461 — TAAGTATTGTATTTGTGTGTGTAT 462 — TGTATTAGTAAAGAAGAGAGAATA 463 — GAGAAGAGAAATAAGTTGAAATAA 464 — GTAAAGTAGAAATAGAATTGAGTT 465 — GTGTGTTATTTGTTTGTAAAGTAT 466 69 TTTGATGTATGAATATAGTATGAG 467 — AAGATTGTGTGAATAGTTGAAATT 468 — TATAAAGTTTGAAGATGAGTGATA 469 — AGATAAAGAGATTTAAGATGTATG 470 71 GAAGAATTAAGTTGAGAATTAAGA 471 — TAGAGAAATTTGATAAAGAAAGAG 472 — AAAGTTTATGAAGTTATTGAGTAG 473 — AAATAGTGTAAGTAAAGAGATGAT 474 — TATGATGATTTAGTTATAAGAGTG 475 — TAGATAAATGTTATGATGAGTAAG 476 — AGATTGATTGTGATGATTTGTATA 477 — TTAAGAAGAATTGTATATGAGAGT 478 73 GTAGAATGTTTAGAGTTGAATATA 479 — GAGAAATAGTAAGAAGTAAATAGA 480 — ATTGAAGTTGTTATGTGAAGATTT 481 — TAAATGTTGTGTAGAGTAATTAGA 482 — AAATAAGAGTTTGAGAAGTTGTTT 483 — AGTTGTAATAAGAAGTGATTTAAG 484 — GTTAGAATGTATATAGAGTTAGAT 485 74 TTGATATTGAAAGAGAAAGTTATG 486 — TTAAAGAGAGAAATGTTTGATTAG 487 — TGTGAATTTGAGTATTAGTAAGAA 488 — TAATTTGAATGTGAAAGTTGTTAG 489 — ATGTGTTTGAAAGATGATGATTTA 490 — AAGTTATGTTGATATTGAGTGAAA 491 — TAGATAAAGAAGATAGAGATTTAG 492 — GATGAATGTAGATATATGTAATGA 493 — GAAGAATAGTTTATGTAAATGATG 494 — GTAGTATATAGTTAAAGATGAGTT 495 — GTTATTTGTGTATGATTATGATTG 496 — AGAGATTAGAAATTGAGAGAATTA 497 — GTATGATAGAGTTTATAGTGATAA 498 — GTTAGAAAGAATGAAATTGAAGTA 499 — AAGAATGAGAATATAGAGATGAAT 500 — AAAGAGAATAGTGTTTAAGAAGAT 501 — GATGTGTTATTGATAGAAATTAGA 502 — TAGAGTTATAGAGATATTGTATGA 503 — GAGAGTTGAATAAGTTAAAGATAT 504 — AGATATGAAATAGATTGTTAGAGA 505 — GAGTGAATAGAAAGATATGTTAAT 506 — AAAGAGATATTGAAGAGAATAAAG 507 — GTTATAGAATAAGTTGTAAAGTGT 508 — TGATAGTATGATAATGTGTTTATG 509 — TTTGTTGTTAAGTATGTGATTTAG 510 77 TAAAGTGTTGTGTTAAAGATTAAG 511 — TGTGTTTGATTGATTAATGTTATG 512 — ATTAATGAATGAGTGTTGTAATGT 513 — TAGATGTTTGTGAGTTTGATATTA 514 — GAATGAATAGTAATAGATGATTTG 515 — AATAGTGTGTTGTTATATGATTAG 516 — TAGATTAGAAGATGTTGTGTATTA 517 — AATGTGTGTGTTAAATGAATTTGT 518 — GAATTAAGTATATGAGTGTAGAAA 519 — TTATTGTGTGTAAGTAGTGTAAAT 520 — GTAGTAAAGAGAATTGTTTAGTAT 521 80 AAGTTTGTAAGAAGTAGTTGAATA 522 — AGTTATAGTATAGTAGTATAGAGA 523 — GAAAGAAATGTGTATAGTTTAATG 524 — TTGTGAGTAATGAATGATGTATTA 525 — GTAGAGTTGTAAATAGAGAATAAA 526 — ATTAATGTAGATTGTAAGAGATAG 527 — TTAGTGTGTTTGTAGATAGAATTA 528 — AGAGAGTTTGTGTATATGTATAAA 529 81 TTAAGTTTAGTGAGATTTGTTAAG 530 — ATGAAGTTTATTGAATAGTAGTGA 531 — ATATTTGTGTTGTATGTTTGTGAA 532 — AAAGTGTTTATAGAAGATTTGATG 533 — AAGAGATATGATTTGTTAGTTGTA 534 — AAGAAGAAATGAGTGATAATGTAA 535 — TAGTGTTTGATATGTTAAGAAGTT 536 — GTAGAAAGTGATAGATTAGTAATA 537 — GATAAATGTTAAGTTAGTATGATG 538 — AGATTAGAAGAATTGTTTAGAATG 539 — ATATTTGAGAAGTGTGAAATGAAT 540 — TGAGTAAATAGTTTATGAGTAGTA 541 — TTAGAGAGTAGATAAAGATTTGAT 542 — ATTGTTTAAGTTGTTGATAAGATG 543 — GTTGTAAAGTTAAAGTGTGAATTT 544 — ATAGATTGTGTGTTTGTTATAGTA 545 — GTAAGTTATTGAGAATGATAATAG 546 — TAGATTAGTTGATAAGTGTGTAAT 547 83 AAATGTAAATGAAGAGTGTTTGTT 548 — GATAGAAGAAATGTATATAGTGAT 549 — TATAGAGTGTATGTTATGATAAAG 550 — TATGAAGTGATAAGATGAAGAATT 551 — TGTTGAGAATAGTAAGAGAATTTA 552 — TAGATAATGTGAAGTAATAAGTGA 553 84 GTATTATGATGATAGTAGTAAGTA 554 — AGATATGATTTAGTATTGAATGTG 555 — AATTAAGTTTGTAGAGTGATTTGA 556 — AAGAAATAGATGTAGTAAGATGTT 557 — TTGAGAAGTTGTTGTAATAAGAAT 558 — AGTGTGAAATAGTGAAAGTTTAAA 559 — TTTATGTAGTAGATTTATGTGAAG 560 — ATTAATGAGAAATTAGTGTGTTAG 561 — ATGTTAATAGTGATAGTAAAGTGA 562 — TATGTTGATAAATGATTATGAGTG 563 — TTATTAGAGTTGTGTGTGATATAT 564 — TGTTGTTATGATTGAGTTAGAATA 565 — AATTTGAGTTAAGAAGAAGTGTAA 566 — AAAGATAAAGTTAAGTGTTTGTAG 567 88 TGTTGAGATGATATTGTATAAGTT 568 — TAAATAGTGAATGAGTTATAGAGT 569 — ATAGATGTTATGATAGTTAGTTAG 570 — GTTAAGTGAAGATATGTATTGTTA 571 — TAAGAAAGTAAAGTTTGTAGATGT 572 — AAGAGAAAGTTTGATTGAATAAAG 573 — ATATTAGATGTGAGTTATATGTGT 574 — AGTTTGAGTTTAGTATTGTGAATA 575 — ATGTTAAATGAGAGATTGTGTATA 576 — TAAATGTTGTGATTATTGTGAGAT 577 — TAAGAATTGAAGTAAGAGTTATTG 578 — AGAGATAGAATTAAGTTTGTTGAT 579 — GAAGAATGTTAAGAAATATGTAAG 580 — TATTTGTGATTAAGAAGTTGAGAA 581 — AGTTAGAATTTGTGTAGTAGAATT 582 — AAGTTTATTGTTGATGTTGTATTG 583 — GAATGAGTTTAAGAGTTTATAGTA 584 — AGTGAAGATTGTATGTAGTATAAA 585 — AGTTGAAATGAGTATTAAGTAATG 586 — ATGTGTTATTTGAGATGAGTAATT 587 — AAATAGTGTTGTTGAAGTTGTTAT 588 — GTAGAGAAAGATATATGTAGTTTA 589 — GAGAGTATTTGATGAATGATTATA 590 — GAGTATAAGTTTAGTGTATATTGA 591 — ATAATGTGATTATTGATTGAGAGA 592 — TTAGTTGTTATGTGAGAGTAATAA 593 — AAATGAGTATATTGAATTGTGATG 594 — AATTAGAAGTAAGTAGAGTTTAAG 595 3 TGTAAGTTTAAAGTAAGAAATGTG 596 5 GAAATGATAAGTTGATATAAGAAG 597 — AATGAGTAGTTTGTATTTGAGTTT 598 — AGTGAATGTAAGATTATGTATTTG 599 6 GTAATTGAATTGAAAGATAAGTGT 600 8 TATGTTTAAGTAGTGAAATAGAGT 601 — GTATTGAAATTGAATTAGAAGTAG 602 — AATATGTAATGTAGTTGAAAGTGA 603 — TGAATATTGAGAATTATGAGAGTT 604 — TAGTGTAAATGATGAAGAAAGTAT 605 — GTATGTGTAAAGAAATTTGATGTA 606 — AATTGTTTGAAAGTTTGTTGAGAA 607 — AATTGTTTGAGTAGTATTAGTAGT 608 — TAATTGAGTTTGAATAAGAGAGTT 609 — TGTTGATTGTAAGTGTTTATTGTT 610 — GAAATTTGTGAGTATGTATTTGAA 611 — TAAGAATGAATGTGAAGTGAATAT 612 — TAATGTGAAGTTTGTGAAAGATAT 613 — TTGTATATGAAAGTAAGAAGAAGT 614 — TAGAGAGAAGAAGAAATAAGAATA 615 — ATTTGAAATGTTAATGAGAGAGAT 616 — TTGTGTGTATATAGTATTAGAATG 617 — ATTGTTAGTATTGATGTGAAGTTA 618 — TGTTTGTATTTGAATGAAATGAAG 619 — TGTTAGATTGTGTTAAATGTACTT 620 — TATAGAGTATTGTATAGACAGAAA 621 — AAATAGTAAGAATGTACTTGTTGA 622 — TGAGTGTGATTTATCATTAAGTTA 623 — ACAATTTGTTGTACTGTTATGATT 624 — GATTGAAGAAAGAAATAGTTTGAA 625 — GATAATAGAGAATAGTAGAGTTAA 626 — GATTGAAATTTGTAGTTATAGTGA 627 — GATTTAAGAAGATGAATAATGTAG 628 — TTTGAGAGAAAGTAGAATAAGATA 629 — GATTAAGAGTAAATGAGTATAAGA 630 — TTTGATAGAATTGAAATTTGAGAG 631 — TGAAGAAGAGTGTTATAAGATTTA 632 — GTGAAATGATTTAGAGTAATAAGT 633 — AAATAAGAATAGAGAGAGAAAGTT 634 9 GTTGTAAAGTAATAGAGAAATTAG 635 — AGTGATTTAGATTATGTGATGATT 636 — AGAGTATAGTTTAGATTTATGTAG 637 — ATGATTAGATAGTGAAATTGTTAG 638 — ATGAAATGTATTAGTTTAGAGTTG 639 — ATATTGAGTGAGAGTTATTGTTAA 640 — AGATGTGTATTGAATTAAGAAGTT 641 — TAATGTGTTGATAGAATAGAGATA 642 — AAATTAGTTGAAAGTATGAGAAAG 643 11 TTTAGAGTTGAAGAAATGTTAATG 644 — GATTGTTGATTATTGATGAATTTG 645 — TGTTGTTGTTGAATTGAAGAATTA 646 — ATTAAGTAAGAATTGAGAGTTTGA 647 12 GTATGTTGTAATGTATTAAGAAAG 648 15 TAGTTGTGATTTATGTAATGATTG 649 — TGATAATGAAAGTTTATAGAGAGA 650 — GTAAGATTGTTTGTATGATAAGAT 651 — TTGAATTAAGAGTAAGATGTTTAG 652 — AAGTGTTTGTTTAGAGTAAAGATA 653 — AGAGAGATAAAGTATAGAAGTTAA 654 — ATTATGAATAGTTAGAAAGAGAGT 655 — TTGTTGATATTAGAGAATGTGTTT 656 — TTTATTGAGAGTTTGTTATTTGTG 657 — AGTGTTAAGAAGTTGATTATTGAT 658 — GAGAAATGATTGAATGTTGATAAT 659 — GATAAGTATTAGTATGAGTGTAAT 660 — TTTGATTTAAGAGTGTTGAATGTA 661 16 AAGTTAGTAAATAGAGTAGAAAGA 662 — GTAAAGTATGAATATGTGAAATGT 663 — TAATAAGTGTGTTGTGAATGTAAT 664 — AAAGATTTAGAGTAGAAAGAGAAT 665 — TTAGTTTGAGTTGAAATAGTAAAG 666 — TAATAGTATGAGTAAGATTGAAAG 667 — GAAGATTAGATTGATGTTAGTTAA 668 — TAAAGAGAGAAGTTAGTAATAGAA 669 — TAAGTATGAGAAATGATGTGTTAT 670 — GAGTTTGTTTGTTAGTTATTGATA 671 — AAGTAAAGAAATGTTAAGAGTAGT 672 — ATGAGAATTGTTGTTGAAATGTAA 673 — TTAGATTAGAGTAGTAGAAGAATA 674 — TAGTGATGAAGAAGTTAGAAATTA 675 — TAATGTAGTAATGTGATGATAAGT 676 — TTGAGAAAGAATAAGTAGTGTAAA 677 — TAATGAGTGAGATTATAGATTGTT 678 — GTATAAGAAATGTGTGTTTGATTA 679 — GTGAATGTGTTAATGAAGATATAT 680 — GAAAGTTATTAGTAGTTAAAGATG 681 — TAGAATTGTGTTTGATAAGTGATA 682 — TGATTTAGATTGAGAGTTAAATGA 683 — ATTATTGAGTTTGAATGTTGATAG 684 — ATAGTAGTTATGTTTGATTTAGTG 685 — ATAGAAGAAGAATAAAGTTAGAGA 686 — GATGTTGAAAGTAATGAATTTGTA 687 — GAGATTGATAGTAGAAATGATAAA 688 — TGAGAGAATAAAGTATGAATTTGA 689 — TATAAAGATGATGTGAATTAGTAG 690 — TTATGTAAGAATGTTTGAGAGAAA 691 — AGTAAATGATGAATGATATGATGA 692 — GAAATTTGTGTTAAAGTTGAATGA 693 — GATGAATGATTGTGTTTAAGTATA 694 — GAAATAAGTGAGAGTTAATGAAAT 695 — TGTTGAAATAGTTATTAGTTTGTG 696 — TTTGAGAGTATATTGATATGAGAA 697 — ATTGTGTGTAAAGTAAGATTTAAG 698 — TATAGTTTGAAGTGTGATGTATTT 699 — GTGAAGTTATAGTGTATAAAGAAT 700 — GTATGTTGAATAGTAAATAGATTG 701 — TTAGAAAGTGTGATTTGTGTATTT 702 — TTTAGTAATATGTAAGAGATGTGA 703 — AGTATGTATAGATGATGTTTGTTT 704 — ATTTAAGTAAAGTGTAGAGATAAG 705 20 ATTTGTGTTGAATTGTAAAGTGAA 706 — ATGTTATTAGATTGTGATGAATGA 707 — TAGTAGTAGAATATGAAATTAGAG 708 — TTTAATGAGAAGAGTTAGAGTATA 709 — AAAGTTTAGTAGAGTGTATGTAAA 710 — ATATATGATAGTAGAGTAGATTAG 711 — TGAGAAGTTAATTGTATAGATTGA 712 — TATAGAGATGTTATATGAAGTTGT 713 — AAATTTGTTAAGTTGTTGTTGTTG 714 — TTGTTGAAGATGAAAGTAGAATTA 715 — AAGAGATAAGTAGTGTTTATGTTT 716 — AATAAGAAGAAGTGAAAGATTGAT 717 — TAAGTTAAAGTTGATGATTGATAG 718 — ATATAAGATAAGAGTGTAAGTGAT 719 — GTTAAATGTTGTTGTTTAAGTGAT 720 — GAGTTAAGTTATTAGTTAAGAAGT 721 — TATTAGAGTTTGAGAATAAGTAGT 722 21 TAATGTTGTTATGTGTTAGATGTT 723 — GAAAGTTGATAGAATGTAATGTTT 724 — TGATAGATGAATTGATTGATTAGT 725 — ATGATAGAGTAAAGAATAAGTTGT 726 — AGTAAGTGTTAGATAGTATTGAAT 727 27 ATGTAGATTAAAGTAGTGTATGTT 728 — TTATTGATAATGAGAGAGTTAAAG 729 — ATTTGTTATGATAAATGTGTAGTG 730 29 TTGAAGAAATAAGAGTAATAAGAG 731 — TGTGTAATAAGTAGTAAGATTAGA 732 — ATGAAAGTTAGAGTTTATGATAAG 733 37 ATTAGTTAAGAGAGTTTGTAGATT 734 — TGTAGTATTGTATGATTAAAGTGT 735 — AGTTGATAAAGAAGAAGAGTATAT 736 — GTAATGAGATAAAGAGAGATAATT 737 — TGTGTTGAAGATAAAGTTTATGAT 738 — AAGAAGAGTAGTTAGAATTGATTA 739 — GAATGAAGATGAAGTTTGTTAATA 740 — AAATTGTTGAGATAAGATAGTGAT 741 — TGATTGTTTAATGATGTGTGATTA 742 — ATGAAGTATTGTTGAGTGATTTAA 743 — GTGTAAATGTTTGAGATGTATATT 744 46 AATTGATGAGTTTAAAGAGTTGAT 745 — TTTGTGTAATATGATTGAGAGTTT 746 — GTAGTAGATGATTAAGAAGATAAA 747 — TTTAATGTGAAATTTGTTGTGAGT 748 — GTAAAGAATTAGATAAAGAGTGAT 749 — AATAGTTAAGTTTAAGAGTTGTGT 750 — GTGTGATGTTTATAGATTTGTTAT 751 — GTATAGTGTGATTAGATTTGTAAA 752 49 GTTGTAAGAAAGATATGTAAGAAA 753 — ATATTAGATTGTAAAGAGAGTGAA 754 — GAGTGATATTGAAATTAGATTGTA 755 — TAAGAAGTTAAAGAAGAGAGTTTA 756 — GATGTTAGATAAAGTTTAAGTAGT 757 — GTGATTGTATGAGAAATGTTAAAT 758 — TGATTATTGTAAGAAAGATTGAGA 759 — AAGAATTGTGTAAGTTTATGAGTA 760 — TTGTATTTAGAAGATTTGTAGATG 761 — TATATGTTTGTGTAAGAAGAAATG 762 — GATAATGTGTGAATTTGTGAATAA 763 — TTAGAAATGTGAGATTTAAGAGTT 764 — AGTGTAGAATTTGTATTTAGTTGT 765 — TAGTTAAGATAGAGTAAATGATAG 766 — GAAGTGATATTGTAAATTGATAAG 767 — GTAATTGTGTTAGATTTAAGAAGT 768 — TGATATTTGTGAATTGATAGTATG 769 — AAGTAAAGAGATATAGTTAAGTTG 770 — ATTAGTTAAGTTATTTGTGAGTGA 771 — AGATGAAGTAGTTTATGAATTAGA 772 — TGAGTTAGTTAAGTGATAGTTAAA 773 — TTATTGTAGATTTAGAGAAGATGA 774 — TATTTGTGTTTGTTGATTAGATAG 775 — GTATAATGTGTGTGAAAGTTATAA 776 — TATATGTTGAGTATAAAGAGAGAA 777 — TTAGTTAGTTTAAAGATTGTGAGT 778 — TTTAGAATAAGTGATGTGATGAAA 779 — AGAGTAATGTGTAAATAGTTAGAT 780 — TGTGATAAAGAGAAATTAGTTGTT 781 — GAATTTAGTGAATGTTTGAGATTA 782 — TGTGATGTGTAAGTATATGAAATT 783 — TTGTGAATGATTAATGAATAGAAG 784 51 AATGTTGTTTAGATTGAGAAAGTT 785 — AGATTGTGTTAGTATTAGTATAAG 786 — TTGATGTATTAGAAAGTTTATGTG 787 — TATGATTGTGTGTTAGAGAATTTA 788 — TAGTGTAGATATTTGATAGTTATG 789 52 AGTTTAATGTGTTTAGTTGTTATG 790 — TGTGTAAAGTAGAAAGTAAAGATT 791 — GTTATGATATAGTGAGTTGTTATT 792 53 TTTGATTGAATGTTAATAGTGTGT 793 — AGAGTATTAGTAGTTATTGTAAGT 794 54 TAAGTAGAAAGAAGAAGATATTTG 795 — AGAAAGAGAATTATGTAATGAAAG 796 — TTAGATTTGTTAGTGTGATTTAAG 797 — GATGATTAAGATATAGAGATAGTT 798 — ATATTTGAGTGATTAAGAGTAATG 799 — TGTATTGTGAGTTAAGTATAAGTT 800 — AATTTAGTAGAAAGTGTTGTGTTT 801 — GTTAGAAGATTAAGTTGAATAATG 802 — TAAAGTATGTGAGATGATTTATGT 803 — TGAAATGATTAAAGATGAAGATGA 804 — TTATTAGATGTTGAGTGTTTGTTT 805 — TAGTGTTTAAAGAGTAGTATATGA 806 — AGTTATAAGTAAATGATGTTGATG 807 — TTAAGAGAGAAATAAGTGTATTGT 808 — GATATTGAAATGTGTAAATGATGA 809 — ATGATGAATTAAGAAAGAAAGAGA 810 — GAATAGTTTGATTTGTGTTTGTTA 811 — AGTTGTTTAGATTTGATTTGTAAG 812 — GTATGAGATTTGATATAAGATTAG 813 — TTTATAGTGAGTATAGTGATGATT 814 — TATATGTGAAGATATAAGTGTTTG 815 — ATTGATAGATGATAGTAATTGAGT 816 — TGATAGATGTGAAGAATTTGATTT 817 — GAAGATATTGAAAGAATTTGATGT 818 55 GATGTTTAGTGTAGATATAGATTT 819 — GAATATTGAGTTATAAGTAGTAGT 820 — AGTGAGTAAGTAATAGAAAGATTT 821 — GTAGAATAAGTAATTTGTGAGATA 822 — GAGTTATTTGAGATTTAGATGTTT 823 — GAAATGATGATTGAATTTAGAGAT 824 — AAATAGTGTGAGAATAGTTAAGTA 825 — ATGTGTTAAGTTGTAGAAGAATAA 826 — ATAATGAGTTAATAGTGTAAGAAG 827 — ATAAGAGATGTTTAAGTTAGAAAG 828 — TGTTAGTGTTAGAAATATGAAAGA 829 — TTTAGAAGATTGTTAGATAAGTTG 830 — GTGTAATGTATAAGATAGTTAAGT 831 — TATTAGAGAGAAATTGTAGAGATT 832 57 TAGTGAGATAAAGTAAAGTTTATG 833 — TTGTGAAAGTTAAGTAAGTTAGTT 834 — AAAGTGTAAGTTGAAGAATATTGA 835 — GAATAGAGTGTTATTTGAAATAGA 836 — TATAAGAGAGAGATAAGTAATAAG 837 — TGAGTGAAATTGATAGAGTAAATT 838 — GATGAATAAGTTTAAGTGAGAAAT 839 — GTGTGATATGTTTATTGATTAAGT 840 — TAAAGTGAGTGTAAATGATAATGA 841 — GTAGAGTTTGATTTGAAAGAATAT 842 — GAATATTGTTATGTTTGTTATGAG 843 — GTGTAATAAGATGTATTGTTGTTT 844 — TAAATTGATTGTGAGTTGAAGAAT 845 — TGAGATAGTTATAGTTAAGTTTAG 846 — AGTTTGTTAAGATTATGTAGAAAG 847 — GAATGTGTAGAATAAGAGATTAAA 848 — GTATTATGAAAGAAGTTGTTGTTT 849 — GTGTTATAGAAGTTAAATGTTAAG 850 58 TTAAGAGTAGTGAATATGATAGTA 851 — AATGTTATAAGATGAGAGTTTAGT 852 — ATATAAGATTTGATGTAGTGTAGT 853 — TATGTTTGTTGTTGTTAAGTTTGA 854 — GATAGTTTAGTATAGAAGATAAAG 855 — GTTGAATATAGAGATAGTAAATAG 856 — AGAGAAGATTTAGTAAGAATGATA 857 — TGAATGAGAAAGATATTGAGTATT 858 — TGAAGATTATAGTAGTTGTATAGA 859 — GATTAGTAGTATTGAAGATTATGT 860 — TGAAATGTGTATTTGTATGTTTAG 861 59 ATTAAAGTTGATATGAAAGAAGTG 862 — AATGTAGAGATTGTAGTGAATATT 863 62 TTATTTGTTGAGTGTAAATGTGAT 864 — ATGTAATTGTGAATAATGTATGTG 865 63 GATTTGTATAGAGATTAGTAAGTA 866 — AATATTGTTGTTTAGAGAAAGAAG 867 — ATGATGATGTATTTGTAAAGAGTA 868 — AATGTATTTGTGTGATTGTGTAAA 869 — AGTGTTATGAAGAATAGTAAGAAT 870 — GTTATGTAGAGATGAAAGAAATTA 871 65 GTTTGTATTAGATAAATGAGTTGT 872 — TGATTTATGAGATTAAGAGAAAGA 873 — TTTGTGTGTTATTGTAATTGAGAT 874 70 GATGTGTGATATGATTAAAGAAAT 875 — AGATTATAGATTTGTAGAGAAAGT 876 — GAAGAGTATGTAATAGTATTGTAT 877 — TTTGTAATGTTGTTGAGTTTAAGA 878 — AGTAAATAGTAGTATGAATAAGAG 879 — GAATGTTGAATTGAAATATGAGTT 880 — AGTAGTTAATTGATAGTAAGTTTG 881 — AGTGTAAAGAAATGAATGAATAAG 882 — TGTTAGATATTTGTGAAATGTGAA 883 — TGTATGTTGAGTTTGAATTGTTAT 884 — TGAGTGAATTAGTTATGTTGTTAT 885 — GAAGAAAGAAATGAGAAAGATTAT 886 — TTAAGTAAGTTGTGTTGATATTAG 887 — ATGATGTGTTTGATTTGAATTGAA 888 72 AAGTAAGTGAAATTGTTGTTTGAA 889 — ATGAAGTGTAAAGTTTGAAAGAAA 890 — AGAGAGTAAGATAATTGTATAGTA 891 — TTTATGAGATAGATGAAATAAGTG 892 — AGAAATTAGTAGTAATGATTTGTG 893 — GATTTGAGATTGAATGAGAATATA 894 — GATTAGAAAGATGAATAAAGATGA 895 — TAGATAGAAAGTATATGTTGTAGT 896 — GAAGATAGTAAAGTAAAGTAAGTT 897 — AAATGTGTGTTTAGTAGTTGTAAA 898 75 TTGTTGAAGTAAGAGATGAATAAA 899 — TATTTGAGAGAAAGAAAGAGTTTA 900 — TATTTAGTGATGAATTTGTGATGT 901 — TTATAGTGATGATGATAAGTTGAT 902 — TAAAGATAATTGTAGAAAGTAGTG 903 — GTTTAGTATTGATATTGTGTGTAA 904 — GTGTTGTGAATAAGATTGAAATAT 905 — AAAGAAAGTATAAAGTGAGATAGA 906 — TATTTGTAAGAAGTGTAGATATTG 907 — TAGAAGATGAAATTGTGATTTGTT 908 — ATAATAGTAAGTGAATGATGAGAT 909 — AATGTGAATAAGATAAAGTGTGTA 910 — ATTGAAGATAAAGATGTTGTTTAG 911 — TGAAATAGAAGTGAGATTATAGTA 912 76 AGTTATTGTGAAAGAGTTTATGAT 913 — AAATAGTAGTGATAGAGAAGATTT 914 — AGTGTATGAAGTGTAATAAGATTA 915 — TGATTAAGATTGTGTAGTGTTATA 916 — AGTTTATGATATTTGTAGATGAGT 917 — TATGTGTATGAAGATTATAGTTAG 918 78 GAAATTGTTGTATAGAGTGATATA 919 — TAGAAATAGTTTAAGTATAGTGTG 920 — TGATTTAGATGTTTATTGTGAGAA 921 — AAGTTGATATTTGTTGTTAGATGA 922 — TGATGTGATAATGAGAATAAAGAA 923 79 AAAGTTTAGTTTGTATTAGTAGAG 924 — AGTTTGATGTGATAGTAAATAGAA 925 — AAGTGTTATTGAATGTGATGTTAT 926 — AAATTGAAGTGTGATAATGTTTGT 927 — GTTTAGTGATTAAAGATAGATTAG 928 82 ATAAGTGTATAAGAGAAGTGTTAA 929 — ATGAATTTGTTTGTGATGAAGTTA 930 — AAAGAATTGAGAAATGAAAGTTAG 931 — AGTGTAAGAGTATAAAGTATTTGA 932 — GAATTAAGATTGTTATATGTGAGT 933 — TATGAAAGTGTTGTTTAAGTAAGA 934 — TAAAGTAAATGTTATGTGAGAGAA 935 — AAAGATATTGATTGAGATAGAGTT 936 — AAGTGATATGAATATGTGAGAAAT 937 — AAATAGAGTTTGTTAATGTAAGTG 938 — GATTTAGATGAGTTAAGAATTTAG 939 — TTGTAAATGAGTGTGAATATTGTA 940 — AGTAGTGTATTTGAGATAATAGAA 941 — TGAGTTAAAGAGTTGTTGATATTT 942 — AAAGAGTGTATTAGAAATAGTTTG 943 — GTTTAGTTATTTGATGAGATAATG 944 — AAGTGTAAATGAATAAAGAGTTGT 945 — AATAAAGTGAGTAGAAGTGTAATT 946 — TATTGAGTTTGTGTAAAGAAGATA 947 — TTTATAGTTGTTGTGTTGAAAGTT 948 — ATGAAATATGATTGTGTTTGTTGT 949 — AAAGAGATGTAAAGTGAGTTATTA 950 — TTGAAGAAAGTTAGATGATGAATT 951 — ATGTTATTTGTTTAGTTTGTGTGA 952 — AAATATGAATTTGAAGAGAAGTGA 953 — GATTAGATATAGAATATTGAAGAG 954 — TTAGAATAAGAGAAATGTATGTGT 955 — TTTATGAAAGAGAAGTGTATTATG 956 — GTAAGTATTAAGTGTGATTTAGTA 957 — ATAAAGAGAAGTAAAGAGTAAAGT 958 — ATTGTTAATTGAAGTGTATGAAAG 959 — TATATAGTTGAGTTGAGTAAGATT 960 — TAGATGAGATATATGAAAGATAGT 961 — ATAAGAAGATGATTTGTGTAAATG 962 — TTAGTAATAAGAAAGATGAAGAGA 963 — GATTTGTGAGTAAAGTAAATAGAA 964 — AAATAGATGTAGAATTTGTGTGTT 965 — GAAATTAGTGTTTGTGTGTATTAT 966 — ATTTGAGTATGATAGAAGATTGTT 967 — ATAGAGTTGAAGTATGTAAAGTTT 968 — TAATTTGTGAATGTTGTTATTGTG 969 — TTAGTTTATGAGAGTGAGATTTAA 970 — GTTGTTAGAGTGTTTATGAAATTT 971 — TTTATTGTGATGTGAAATAAGAGA 972 — GTAAGTAATATGATAGTGATTAAG 973 — TGAGATGATGTATATGTAGTAATA 974 — AATTGAGAAAGAGATAAATGATAG 975 85 TTTGAAGTGATGTTAGAATGTTTA 976 — AGTTGTTGTGTAATTGTTAGTAAA 977 — ATAGTGAGAAGTGATAAGATATTT 978 — GTGTGATAAGTAATTGAGTTAAAT 979 — TAGTTATTGTTTGTGAATTTGAGA 980 — ATAGTTGAATAGTAATTTGAAGAG 981 — ATGTTTGTGTTTGAATAGAGAATA 982 — TGATAAAGATATGAGAGATTGTAA 983 — TAAAGATGAGATGTTGTTAAAGTT 984 — AAGTGAAATTTGTAAGAATTAGTG 985 — GAAATGAGAGTTATTGATAGTTTA 986 — TTTGTAAATGAGATATAGTGTTAG 987 — GTTAATTGTGATATTTGATTAGTG 988 — AGAGTGTTGATAAAGATGTTTATA 989 — AATTGTGAGAAATTGATAAGAAGA 990 — TTAAAGAGAATTGAGAAGAGAAAT 991 — TTGTTAGAAGAATTGAATGTATGT 992 — AGTTAAGATATGTGTGATGTTTAA 993 — TGAGTTATGTTGTAATAGAAATTG 994 — TTAGATAAGTTTAGAGATTGAGAA 995 — ATGAGTAATAAGAGTATTTGAAGT 996 — TGTTTAAGTGTAATGATTTGTTAG 997 — TTGAAGAAGATTGTTATTGTTGAA 998 — TATAGAAAGATTAAAGAGTGAATG 999 — TAAATTGTTAGAAATTTGAGTGTG 1000 — ATTGTTAGTGTGTTATTGATTATG 1001 — GAGAATTATGTGTGAATATAGAAA 1002 — TTGATTGATAAAGTAAAGAGTGTA 1003 — GTGTGTAAATTGAATATGTTAATG 1004 — AAAGTAAAGAAAGAAGTTTGAAAG 1005 — TTTAGTTGAAGAATAGAAAGAAAG 1006 — GTGTAATAAGAGTGAATAGTAATT 1007 — TATTGAAATAAGAGAGATTTGTGA 1008 — ATGAGAAAGAAGAAGTTAAGATTT 1009 — AAGAGTGAGTATATTGTTAAAGAA 1010 — TTTGTAAAGTGATGATGTAAGATA 1011 — GATGTTATGTGATGAAATATGTAT 1012 — GTAGAATAAAGTGTTAAAGTGTTA 1013 — AAAGAGTATGTGTGTATGATATTT 1014 — AAAGATAAGAGTTAGTAAATTGTG 1015 — AAGAATTAGAGAATAAGTGTGATA 1016 — GATAAGAAAGTGAAATGTAAATTG 1017 86 GATGAAAGATGTTTAAAGTTTGTT 1018 — AGTGTAAGTAATAAGTTTGAGAAA 1019 — GTTGAGAATTAGAATTTGATAAAG 1020 87 TTAAGAAATTTGTATGTGTTGTTG 1021 — AGAAGATTTAGATGAAATGAGTTT 1022 — TAAGTTTGAGATAAAGATGATATG 1023 — TGAGATAGTTTGTAATATGTTTGT 1024 — AGTTTGAAATTGTAAGTTTGATGA 1025 — TAGAATTGATTAATGATGAGTAGT 1026 — AGAGATTTGTAATAAGTATTGAAG 1027 — ATAATGATGTAATGTAAGTAGTGT 1028 — TGAAATTTGATGAGAGATATGTTA 1029 — TGTGTAAAGTATAGTTTATGTTAG 1030 — TGAATAAGTGAAATAGAATGAATG 1031 — AAAGAAAGATTGTAATAAGTAGAG 1032 — AATGAAATAGTGTTAAATGAGTGT 1033 89 GTAGATAAAGATGTGAATTATGAT 1034 — GATAGTATATGTGTGTATTTGTTT 1035 — ATGTTTGTAGAAATGTTTGAAGAT 1036 — AAATTTGTAGAGAGAAATTTGTTG 1037 — TAGAATAAGATTAGTAAGTGTAGA 1038 — TGATTTAGAGAAATATGAGTAGAA 1039 — AATAGAGTATGTTGTTTATGAGAA 1040 — GATGATGAAGAGTTTATTGTAAAT 1041 — AAGTAAAGAAGAAGAAATGTGTTA 1042 — TTGAAGAATTAAGTGTTTAGTGTA 1043 — AGAAAGAATGTTGATTTATGATGT 1044 — GATTAAAGAGATGTTGATTGAAAT 1045 — AATGATAATTGTTGAGAGAGTAAT 1046 — GTTTGTTGAAAGTGTAAAGTATAT 1047 90 TGAGTTATATGAGAAAGTGTAATT 1048 — TTGTGAGAAAGAAGTATATAGAAT 1049 — GTAAGTTTAGAGTTATAGAGTTTA 1050 — GATAGATAGATAAGTTAATTGAAG 1051 — AGAGATGATTGTTTATGTATTATG 1052 — AAAGTTAAGAAATTGTAGTGATAG 1053 — TTTGATATTGTTTGTGAGTGTATA 1054 — ATTTGTAGAAAGTTGTTATGAGTT 1055 — GATTTGAGTAAGTTTATAGATGAA 1056 — AAGATAAAGTGAGTTGATTTAGAT 1057 — GATATTGTAAGATATGTTGTAAAG 1058 — GTAAGAGTGTATTGTAAGTTAATT 1059 — GTGTGATTAGTAATGAAGTATTTA 1060 91 GTAAGAAAGATTAAGTGTTAGTAA 1061 — AGTAGAAAGTTGAAATTGATTATG 1062 92 TAAGAGAAGTTGAGTAATGTATTT 1063 — GTTAAGAAATAGTAGATAAGTGAA 1064 — TAAGTAAATTGAAAGTGTATAGTG 1065 — AAGATGTATGTTTATTGTTGTGTA 1066 — ATTTAGAATATAGTGAAGAGATAG 1067 — GTTATGAAAGAGTATGTGTTAAAT 1068 93 TATTATGTGAAGAAGAATGATTAG 1069 — TAATAAGTTGAAGAGAATTGTTGT 1070 — TGATGTTTGATGTAATTGTTAAAG 1071 — GTGAAAGATTTGAGTTTGTATAAT 1072 — AGAGAATATAGATTGAGATTTGTT 1073 — TTTGAGATGTGATGATAAAGTTAA 1074 — GTTGTAAATTGTAGTAAAGAAGTA 1075 94 GTGTTATGATGTTGTTTGTATTAT 1076 — ATTATTGTGTAGATGTATTAAGAG 1077 — GTTAGAAAGATTTAGAAGTTAGTT 1078 — TTGTGTATTAAGAGAGTGAAATAT 1079 — GTTTAAGATAGAAAGAGTGATTTA 1080 — AATGAGAAATAGATAGTTATTGTG 1081 — TGAATTGAATAAGAATTTGTTGTG 1082 95 AATAAGATTGAATTAGTGAGTAAG 1083 — AATGTTTGAGAGATTTAGTAAAGA 1084 — AGTTTAGAATAGAAATGTGTTTGA 1085 — TATAAGTAAGTGTTAAGATTTGAG 1086 — GTAGTGAATAAGTTAGTGTTAATA 1087 — AAGTGTGTTAAAGTAAATGTAGAT 1088 — AGAGATGTTTATGTTGTGAATTAA 1089 — AGTTGAATATTGATGATAAGAAGA 1090 — TGAATGTGAGATGTTTAGAATAAT 1091 — AATAATGATGTAAGTTTGAGTTTG 1092 — AAAGAGTGAATAGAAATAAGAGAA 1093 — AATAAAGTTATTGAGAGAGTTTAG 1094 — AGTAGTGTTGTAGTTTAGTATATA 1095 — GTAAGAATGTATTAGATATTTGTG 1096 — GATAAATGTTTGATAAAGTAGTTG 1097 — ATAGTATGTATGTGTGAAGATTTA 1098 — ATGAATGTAGAGTGATTAGTTTAA 1099 — GTAGTATTTAGTGATGTAAGAATA 1100 — AGAATTGTATTGAAGAAGAATATG 1101 — TTTATAGAATTGAGAGAAGTTAAG 1102 — AAAGTAGTAGAGATTTGAGAATTA 1103 — TTTAAAGAAAGTATTGTAAGAGTG 1104 — AAATTGAGAAAGTGAATGAAGTTT 1105 — AAGAAATAAGTATGATAGTAGTAG 1106 — ATTTGAATTGTATTGTAGTTTGTG 1107 — AAGAGAATAATGTAGAGATATAAG 1108 — TGTGTAATAGTTGTTAATGAGTAA 1109 — TATAGTTGTAGTTTAGATGAATGT 1110 — ATTGTGTTAGAATGATGTTAATAG 1111 — GTTTGTATAGTATTTGATTGATGT 1112 — AGAGTAAAGTATGAGTTATGAATA 1113 — GAAAGTTTAAGTGATGTATATTGT 1114 96 TTAAATGATAAAGAGTAGTGAAGT 1115 — TTAAATGTGTGAGAAGATGAATAA 1116 — ATTTGTATAAAGTGAAGAAGAGAA 1117 97 TGATTAGTATTTGTGAAGAGATTT 1118 — TTTGAATGAAATTGATGATAGATG 1119 — AGAGTAAGATTAAGAATAAGAAAG 1120 — ATTGAATTGAGAAGTGAAGTAAAT 1121 — TTTAGAGAAGTATTGTTTGAAAGA 1122 — TAAAGTGAAAGATTTGAAATGATG 1123 — GAAAGTTAGAGAAATGTAGAAATT 1124 — GTGAATAATGAAGAAGTTATGTTA 1125 98 TTGTGAATAAAGTAGATGTGTTAT 1126 — TTATATGATATGAGTTTGTGTTGA 1127 — TTGATTTGTGTGAGTATTAGTTAT 1128 — AAAGTGATTAAGTTAGTTTGAGAT 1129 — TTGTATTTGTATAATGTTGAAGAG 1130 — GTTTGAAATTAGTGTGAGAAATAT 1131 — AATGTTGAGATTGATAATGTTGAA 1132 — TAGTAGTAGTATTGTTGTAATAAG 1133 — GTTGTAATTTGAGTGTTAGTTATT 1134 — TGAATATGATAGTTAGTAATTGTG 1135 — TGATAGTATGTTTGTGATTAAAGA 1136 — GATGTATAAAGAGTATGTTATAAG 1137 — AGTGAGATTTAGAAGATGTTATTA 1138 — ATGAGAATTTGTTAAAGAGAAAGT 1139 — AAAGAATTAGTATGATAGATGAGA 1140 99 TAGAGTTGTATAGTTTATAGTTGA 1141 — GTAGAATGATTGTTTAGAAGATTT 1142 — GTTTATGTTTGAGAAGAGTTATTT 1143 — TAGAAGTTTGAAAGTTATTGATTG 1144 — GATGAAGAGTATTTGTTATATGTA 1145 — GATGAATATAGTAAGTATTGAGTA 1146 100 TAGTGATGAAATTTGAGATAGATA 1147 — GAAAGAAATTGAAGAGTTTGATAT 1148 — ATTTGAGTATTTGTGTATTGAATG 1149 — ATGAGTTGAAATTTGAAGTATTGT 1150 — TTAATAGTGAGAGAGTATATGTAA 1151 — ATTAAGAGAGTGAGTAAATGTAAA 1152 — AAGAATAGATGAGATTAGAAATAG 1153 — AGTTTAAAGAGTTAGAATTGAAAG 1154 — GTAAGATTTGTTGAATAAAGAAGA 1155 — AGAGAAAGAAGTTAAAGTGATATT 1156 — TAATAGAGAAGAGATGTATGAATA 1157 — TTATTAGTGATAAGTGAAGTTTAG 1158 — ATAATGTAAAGATGAGTTTATGAG 1159 — TTGATTTGAGAGTTGATAAGATTT 1160 — ATGATTATTGTGTGTAGAATTAGA 1161 — TATAAAGATATAGTAGATGATGTG 1162 — TTTAGTTGAGATGAAGTTATTAGA 1163 — ATTGAATTGATATAGTGTAAAGTG 1164 — GAAGAAAGATTATTGTATTGAGTT 1165 — ATTGAGTGTAGTGATTTAGAAATA 1166 — AATAAAGTGTTTAAGAGTAGAGTA 1167 — GTAGAGATAATTGATGTGTAATTT 1168 — 

1. A composition comprising a cleavage structure, said cleavage structure comprising: a) a target nucleic acid having a first region, a second region and a third region, wherein said first region is located adjacent to and downstream from said second region and said second region is located to and downstream from said third region; b) a first oligonucleotide having a 5′ portion and a 3′ portion, said 5′ portion of said first oligonucleotide having a sequence complementary to said second region of said target nucleic acid and said 3′ portion of said first oligonucleotide having a sequence complementary to said third region of said target nucleic acid; and c) a second oligonucleotide having a 5′ portion, a central portion and a 3′ portion, said 5′ portion of said second oligonucleotide having a sequence complementary to said first region of said target nucleic acid, said central region of said second oligonucleotide having a sequence complementary to said second region of said target nucleic acid, and said 3′ portion of said second oligonucleotide having a sequence that is not base-paired to said target nucleic acid and is selected from a set of oligonucleotides based on a following group of sequences, 1 4 6 6 1 3 2 4 5 5 2 3 1 8 1 2 3 4 1 7 1 9 8 4 1 1 9 2 6 9 1 2 4 3 9 6 9 8 9 8 10 9 9 1 2 3 8 10 8 8 7 4 3 1 1 1 1 1 1 2 2 1 3 3 2 2 3 1 2 2 3 2 4 1 4 4 4 2 1 2 3 3 1 1 1 3 2 2 1 4 3 3 3 3 3 4 4 3 1 1 4 4 3 4 1 1 3 3 3 6 6 6 3 5 6 6 1 1 6 5 7 6 7 7 7 5 8 7 5 5 8 8 2 1 7 7 1 1 2 3 2 3 1 3 2 6 5 6 1 6 4 8 1 1 3 8 5 3 1 1 6 3 5 6 8 8 6 6 8 3 6 5 7 3 1 2 3 1 4 6 1 5 7 5 4 3 2 1 6 7 3 6 2 6 1 3 3 1 2 7 6 8 3 1 3 4 3 1 2 5 3 5 6 1 2 7 3 6 1 7 2 7 4 6 3 5 1 7 5 4 6 3 8 6 6 8 2 3 7 1 7 1 7 8 6 3 7 3 4 1 6 8 4 7 7 1 2 4 3 6 5 2 6 3 1 4 1 4 6 1 3 3 1 4 8 1 8 3 3 5 3 8 1 3 6 6 3 7 7 3 8 6 4 7 3 1 3 7 8 6 10 9 5 5 10 10 7 10 10 10 7 9 9 9 7 7 10 9 9 3 10 3 10 3 9 6 3 4 10 6 10 4 10 3 9 4 3 9 3 10 4 9 9 10 5 9 4 8 3 9 4 9 10 7 3 5 9 4 10 8 4 10 5 4 9 3 5 3 3 9 8 10 6 8 6 9 7 10 4 6 10 9 6 4 4 9 8 10 8 3 7 7 9 10 5 3 8 8 9 3 9 10 8 10 2 9 5 9 9 6 2 2 7 10 9 7 5 3 10 6 10 3 6 8 9 2 10 9 3 2 7 3 8 9 10 3 6 2 3 2 5 10 8 9 8 2 3 10 2 9 6 3 9 8 2 10 3 7 3 9 9 10 9 10 1 1 9 4 10 1 9 1 4 1 7 1 10 9 8 1 9 1 10 1 10 6 9 6 9 1 3 10 3 10 8 8 9 1 3 8 1 9 10 3 9 10 1 3 6 9 1 9 1 10 3 1 1 4 9 6 8 10 3 3 9 6 1 10 5 3 1 6 9 10 6 1 8 10 9 6 5 9 9 4 10 3 2 10 9 1 9 5 10 10 7 2 1 9 10 9 9 1 8 2 1 8 6 8 9 10 1 9 1 3 8 10 9 6 9 10 1 2 1 10 8 9 9 2 1 9 6 7 2 9 4 3 9 3 5 1 5 11 10 14 12 1 7 12 4 13 3 2 5 5 4 4 12 9 2 13 13 11 13 13 10 2 5 4 12 7 11 7 4 11 6 4 12 12 1 9 11 11 12 9 4 14 12 6 12 7 13 2 9 11 9 11 3 4 1 3 10 5 12 11 4 4 4 13 7 12 1 5 9 13 10 11 11 6 10 14 14 10 1 3 2 14 1 10 4 5 10 12 12 7 11 10 9 11 2 12 8 11 2 8 5 2 12 14 1 8 13 3 7 8 9 4 7 5 4 2 13 2 12 7 1 12 11 10 9 7 5 11 8 12 2 2 12 7 5 2 14 3 4 13 1 8 8 1 5 9 14 5 11 10 13 3 14 1 4 13 2 4 4 4 5 11 3 10 10 9 2 3 3 11 11 4 8 14 3 4 5 1 14 8 11 2 14 3 11 6 12 5 13 4 4 1 10 1 6 10 11 6 5 1 5 8 12 5 1 7 4 5 9 6 9 2 13 2 4 4 2 3 11 2 2 5 9 3 8 1 10 12 2 8 12 7 9 11 4 1 12 1 4 14 3 13 11 2 7 10 4 1 3 4 12 11 11 11 3 3 4 2 12 11 1 5 9 4 2 1 6 1 12 2 10 5 10 5 1 12 2 14 2 11 7 9 4 11 7 4 4 5 14 12 12 5 2 1 10 12 5 9 2 11 6 1 12 14 3 6 1 14 5 9 11 10 1 4 2 5 12 14 10 10 4 5 8 4 5 6 10 12 4 6 12 5 4 2 1 13 6 8 9 10 10 14 5 3 6 14 10 11 3 3 2 9 10 12 5 7 13 3 7 10 5 12 6 4 1 2 5 13 6 1 13 4 14 13 2 12 1 14 1 9 4 11 13 2 6 10 1 10 7 4 5 8 7 2 2 10 13 4 8 2 11 4 6 14 4 8 2 6 2 3 7 1 12 11 2 9 5 6 10 4 13 4 5 10 4 11 9 3 3 11 9 3 2 3 8 15 6 20 17 19 21 10 15 3 7 11 11 7 17 20 14 9 16 6 17 13 21 21 10 15 22 6 17 21 15 7 17 10 22 22 3 20 8 15 20 16 17 21 10 16 6 22 6 21 14 14 14 16 7 17 3 20 10 7 16 19 14 17 7 21 20 16 7 15 22 10 20 10 18 11 22 18 18 7 19 15 7 22 21 18 7 21 16 3 14 13 7 22 17 13 19 7 8 12 10 17 15 3 21 14 9 7 19 6 15 7 14 14 4 17 10 15 20 19 21 6 18 4 20 16 2 19 8 17 6 13 12 12 6 17 4 20 16 21 12 10 19 16 14 14 15 2 7 21 8 16 21 6 22 16 14 17 22 14 17 20 10 21 7 15 21 18 16 13 20 18 21 12 15 7 4 22 14 13 7 19 14 8 15 4 4 5 3 20 7 16 22 18 6 18 13 20 19 6 16 3 13 3 18 6 22 7 20 18 10 17 11 21 8 13 7 10 17 19 10 14

wherein: (A) each of 1 to 22 is a 4mer selected from the group of 4mers consisting of WWWW, WWWX, WWWY, WWXW, WWXX, WWXY, WWYW, WWYX, WWYY, WXWW, WXWX, WXWY, WXXW, WXXX, WXXY, WXYW, WXYX, WXYY, WYWW, WYWX, WYWY, WYXW, WYXX, WYXY, WYYW, WYYX, WYYY, XWWW, XWWX, XWWY, XWXW, XWXX, XWXY, XWYW, XWYX, XWYY, XXWW, XXWX, XXWY, XXXW, XXXX, XXXY, XXYW, XXYX, XXYY, XYWW, XYWX, XYWY, XYXW, XYXX, XYXY, XYYW, XYYX, XYYY, YWWW, YWWX, YWWY, YWXW, YWXX, YWXY, YWYW, YWYX, YWYY, YXWW, YXWX, YXWY, YXXW, YXXX, YXXY, YXYW, YXYX, YXYY, YYWW, YYWX, YYWY, YYXW, YYXX, YYXY, YYYW, YYYX, and YYYY, and (B) each of 1 to 22 is selected so as to be different from all of the others of 1 to 22; (C) each of W, X and Y is abase in which: (i) (a) W=one of A, T/U, G, and C, X=one of A, T/U, G, and C, Y=one of A, T/U, G, and C, and each of W, X and Y is selected so as to be different from all of the others of W, X and Y,  (b) an unselected said base of (i)(a) can be substituted any number of times for any one of W, X and Y, or (ii) (a) W=G or C, X=A or T/U, Y=A or T/U, and X≠Y, and  (b) a base not selected in (ii)(a) can be inserted into each sequence at one or more locations, the location of each insertion being the same in all the sequences; (D) up to three bases can be inserted at any location of any of the sequences or up to three bases can be deleted from any of the sequences; (E) all of the sequences of a said group of oligonucleotides are read 5′ to 3′ or are read 3′ to 5′; and wherein each oligonucleotide of a said set has a sequence of at least ten contiguous bases of the sequence on which it is based, provided that: (F) (I) the quotient of the sum of G and C divided by the sum of A, T/U, G and C for all combined sequences of the set is between about 0.1 and 0.40 and said quotient for each sequence of the set does not vary from the quotient for the combined sequences by more than 0.2; and (II) for any phantom sequence generated from any pair of first and second sequences of the set L₁ and L₂ in length, respectively, by selection from the first and second sequences of identical bases in identical sequence with each other: (i) any consecutive sequence of bases in the phantom sequence which is identical to a consecutive sequence of bases in each of the first and second sequences from which it is generated is less than ((¾×L)−1) bases in length; (ii) the phantom sequence, if greater than or equal to (⅚×L) in length, contains at least three insertions/deletions or mismatches when compared to the first and second sequences from which itis generated; and (iii) the phantom sequence is not greater than or equal to ( 11/12×L) in length; where L=L₁, or if L₁≠L₂, where L is the greater of L₁ and L₂; and wherein any base present may be substituted by an analogue thereof.
 2. The composition of claim 1, wherein one or more of said first and second oligonucleotides contain a 3′-terminal dideoxynucleotide.
 3. The composition of claim 1, wherein the composition includes a plurality of said target nucleic acid sequences and a plurality of second oligonucleotide molecules such that each of said second oligonucleotide molecules has a distinct 3′ region.
 4. The composition of claim 3, wherein the composition includes at least ten said second oligonucleotide molecules or at least eleven said second oligonucleotide molecules, or at least twelve said second oligonucleotide molecules, or at least thirteen said second oligonucleotide molecules, or at least fourteen said second oligonucleotide molecules, or at least fifteen said second oligonucleotide molecules, or at least sixteen said second oligonucleotide molecules, or at least seventeen said second oligonucleotide molecules, or at least eighteen said second oligonucleotide molecules, or at least nineteen said second oligonucleotide molecules, or at least twenty said second oligonucleotide molecules, or at least twenty-one said second oligonucleotide molecules, or at least twenty-two said second oligonucleotide molecules, or at least twenty-three said second oligonucleotide molecules, or at least twenty-four said second oligonucleotide molecules, or at least twenty-five said second oligonucleotide molecules, or at least twenty-six said second oligonucleotide molecules, or at least twenty-seven said second oligonucleotide molecules, or at least twenty-eight said second oligonucleotide molecules, or at least twenty-nine said second oligonucleotide molecules, or at least thirty said second oligonucleotide molecules, or at least thirty-one said second oligonucleotide molecules, or at least thirty-two said second oligonucleotide molecules, or at least thirty-three said second oligonucleotide molecules, or at least thirty-four said second oligonucleotide molecules, or at least thirty-five said second oligonucleotide molecules, or at least thirty-six said second oligonucleotide molecules, or at least thirty-seven said second oligonucleotide molecules, or at least thirty-eight said second oligonucleotide molecules, or at least thirty-nine said second oligonucleotide molecules, or at least forty said second oligonucleotide molecules, or at least forty-one said second oligonucleotide molecules, or at least forty-two said second oligonucleotide molecules, or at least forty-three said second oligonucleotide molecules, or at least forty-four said second oligonucleotide molecules, or at least forty-five said second oligonucleotide molecules, or at least forty-six said second oligonucleotide molecules, or at least forty seven said second oligonucleotide molecules, or at least forty-eight said second oligonucleotide molecules, or at least forty-nine said second oligonucleotide molecules, or at least fifty said second oligonucleotide molecules, or at least sixty said second oligonucleotide molecules, or at least seventy said second oligonucleotide molecules, or at least eighty said second oligonucleotide molecules, or at least ninety said second oligonucleotide molecules, or at least one hundred said second oligonucleotide molecules, or at least one hundred and ten said second oligonucleotide molecules, or at least one hundred and twenty said second oligonucleotide molecules, or at least one hundred and thirty said second oligonucleotide molecules, or at least one hundred and forty said second oligonucleotide molecules, or at least one hundred and fifty said second oligonucleotide molecules, or at least one hundred and sixty said second oligonucleotide molecules, or at least one hundred and seventy said second oligonucleotide molecules, or at least one hundred and eighty said second oligonucleotide molecules, or at least one hundred and ninety said second oligonucleotide molecules, or at least two hundred said second oligonucleotide molecules.
 5. A method of detecting the presence of a target nucleic acid molecule by detecting non-target cleavage products, the method comprising: a) providing: i) a cleavage means, ii) a target nucleic acid, said target nucleic acid having a first region, a second region and a third region, wherein said first region is located adjacent to and downstream from said second region, and said second region is located adjacent to and downstream from said third region; iii) a first oligonucleotide having a 5′ and a 3′ portion, said 5′ portion of said first oligonucleotide having a sequence complementary to said second region of said target nucleic acid and said 3′ portion of said first oligonucleotide having a sequence complementary to said third region of said target nucleic acid; iv) a second oligonucleotide having a 5, portion, a central portion and a 3′ portion, said 5′ portion of said second oligonucleotide having a sequence complementary to said first region of said target nucleic acid, said central portion of said second oligonucleotide having a sequence complimentary to said second region of said target nucleic acid, and said 3′ portion of said second oligonucleotide having a sequence that is not base-paired to said target nucleic acid and is selected from a set of oligonucleotides based one a following group of sequences; 1 4 6 6 1 3 2 4 5 5 2 3 1 8 1 2 3 4 1 7 1 9 8 4 1 1 9 2 6 9 1 2 4 3 9 6 9 8 9 8 10 9 9 1 2 3 8 10 8 8 7 4 3 1 1 1 1 1 1 2 2 1 3 3 2 2 3 1 2 2 3 2 4 1 4 4 4 2 1 2 3 3 1 1 1 3 2 2 1 4 3 3 3 3 3 4 4 3 1 1 4 4 3 4 1 1 3 3 3 6 6 6 3 5 6 6 1 1 6 5 7 6 7 7 7 5 8 7 5 5 8 8 2 1 7 7 1 1 2 3 2 3 1 3 2 6 5 6 1 6 4 8 1 1 3 8 5 3 1 1 6 3 5 6 8 8 6 6 8 3 6 5 7 3 1 2 3 1 4 6 1 5 7 5 4 3 2 1 6 7 3 6 2 6 1 3 3 1 2 7 6 8 3 1 3 4 3 1 2 5 3 5 6 1 2 7 3 6 1 7 2 7 4 6 3 5 1 7 5 4 6 3 8 6 6 8 2 3 7 1 7 1 7 8 6 3 7 3 4 1 6 8 4 7 7 1 2 4 3 6 5 2 6 3 1 4 1 4 6 1 3 3 1 4 8 1 8 3 3 5 3 8 1 3 6 6 3 7 7 3 8 6 4 7 3 1 3 7 8 6 10 9 5 5 10 10 7 10 10 10 7 9 9 9 7 7 10 9 9 3 10 3 10 3 9 6 3 4 10 6 10 4 10 3 9 4 3 9 3 10 4 9 9 10 5 9 4 8 3 9 4 9 10 7 3 5 9 4 10 8 4 10 5 4 9 3 5 3 3 9 8 10 6 8 6 9 7 10 4 6 10 9 6 4 4 9 8 10 8 3 7 7 9 10 5 3 8 8 9 3 9 10 8 10 2 9 5 9 9 6 2 2 7 10 9 7 5 3 10 6 10 3 6 8 9 2 10 9 3 2 7 3 8 9 10 3 6 2 3 2 5 10 8 9 8 2 3 10 2 9 6 3 9 8 2 10 3 7 3 9 9 10 9 10 1 1 9 4 10 1 9 1 4 1 7 1 10 9 8 1 9 1 10 1 10 6 9 6 9 1 3 10 3 10 8 8 9 1 3 8 1 9 10 3 9 10 1 3 6 9 1 9 1 10 3 1 1 4 9 6 8 10 3 3 9 6 1 10 5 3 1 6 9 10 6 1 8 10 9 6 5 9 9 4 10 3 2 10 9 1 9 5 10 10 7 2 1 9 10 9 9 1 8 2 1 8 6 8 9 10 1 9 1 3 8 10 9 6 9 10 1 2 1 10 8 9 9 2 1 9 6 7 2 9 4 3 9 3 5 1 5 11 10 14 12 1 7 12 4 13 3 2 5 5 4 4 12 9 2 13 13 11 13 13 10 2 5 4 12 7 11 7 4 11 6 4 12 12 1 9 11 11 12 9 4 14 12 6 12 7 13 2 9 11 9 11 3 4 1 3 10 5 12 11 4 4 4 13 7 12 1 5 9 13 10 11 11 6 10 14 14 10 1 3 2 14 1 10 4 5 10 12 12 7 11 10 9 11 2 12 8 11 2 8 5 2 12 14 1 8 13 3 7 8 9 4 7 5 4 2 13 2 12 7 1 12 11 10 9 7 5 11 8 12 2 2 12 7 5 2 14 3 4 13 1 8 8 1 5 9 14 5 11 10 13 3 14 1 4 13 2 4 4 4 5 11 3 10 10 9 2 3 3 11 11 4 8 14 3 4 5 1 14 8 11 2 14 3 11 6 12 5 13 4 4 1 10 1 6 10 11 6 5 1 5 8 12 5 1 7 4 5 9 6 9 2 13 2 4 4 2 3 11 2 2 5 9 3 8 1 10 12 2 8 12 7 9 11 4 1 12 1 4 14 3 13 11 2 7 10 4 1 3 4 12 11 11 11 3 3 4 2 12 11 1 5 9 4 2 1 6 1 12 2 10 5 10 5 1 12 2 14 2 11 7 9 4 11 7 4 4 5 14 12 12 5 2 1 10 12 5 9 2 11 6 1 12 14 3 6 1 14 5 9 11 10 1 4 2 5 12 14 10 10 4 5 8 4 5 6 10 12 4 6 12 5 4 2 1 13 6 8 9 10 10 14 5 3 6 14 10 11 3 3 2 9 10 12 5 7 13 3 7 10 5 12 6 4 1 2 5 13 6 1 13 4 14 13 2 12 1 14 1 9 4 11 13 2 6 10 1 10 7 4 5 8 7 2 2 10 13 4 8 2 11 4 6 14 4 8 2 6 2 3 7 1 12 11 2 9 5 6 10 4 13 4 5 10 4 11 9 3 3 11 9 3 2 3 8 15 6 20 17 19 21 10 15 3 7 11 11 7 17 20 14 9 16 6 17 13 21 21 10 15 22 6 17 21 15 7 17 10 22 22 3 20 8 15 20 16 17 21 10 16 6 22 6 21 14 14 14 16 7 17 3 20 10 7 16 19 14 17 7 21 20 16 7 15 22 10 20 10 18 11 22 18 18 7 19 15 7 22 21 18 7 21 16 3 14 13 7 22 17 13 19 7 8 12 10 17 15 3 21 14 9 7 19 6 15 7 14 14 4 17 10 15 20 19 21 6 18 4 20 16 2 19 8 17 6 13 12 12 6 17 4 20 16 21 12 10 19 16 14 14 15 2 7 21 8 16 21 6 22 16 14 17 22 14 17 20 10 21 7 15 21 18 16 13 20 18 21 12 15 7 4 22 14 13 7 19 14 8 15 4 4 5 3 20 7 16 22 18 6 18 13 20 19 6 16 3 13 3 18 6 22 7 20 18 10 17 11 21 8 13 7 10 17 19 10 14

wherein: (A) each of 1 to 22 is a 4mer selected from the group of 4mers consisting of WWWW, WWWX, WWWY, WWXW, WWXX, WWXY, WWYW, WWYX, WWYY, WXWW, WXWX, WXWY, WXXW, WXXX, WXXY, WXYW, WXYX, WXYY, WYWW, WYWX, WYWY, WYXW, WYXX, WYXY, WYYW, WYYX, WYYY, XWWW, XWWX, XWWY, XWXW, XWXX, XWXY, XWYW, XWYX, XWYY, XXWW, XXWX, XXWY, XXXW, XXXX, XXXY, XXYW, XXYX, XXYY, XYWW, XYWX, XYWY, XYXW, XYXX, XYXY, XYYW, XYYX, XYYY, YWWW, YWWX, YWWY, YWXW, YWXX, YWXY, YWYW, YWYX, YWYY, YXWW, YXWX, YXWY, YXXW, YXXX, YXXY, YXYW, YXYX, YXYY, YYWW, YYWX, YYWY, YYXW, YYXX, YYXY, YYYW, YYYX, and YYYY, and (B) each of 1 to 22 is selected so as to be different from all of the others of 1 to 22; (C) each of W, X and Y is a base in which: (i) (a) W=one of A, T/U, G, and C, X=one of A, T/U, G, and C, Y=one of A, T/U, G, and C, and each of W, X and Y is selected so as to be different from all of the others of W, X and Y,  (b) an unselected said base of (i)(a) can be substituted any number of times for any one of W, X and Y, or (ii) (a) W=G or C, X=A or T/U, Y A or T/U, and X≠Y, and  (b) a base not selected in (ii)(a) can be inserted into each sequence at one or more locations, the location of each insertion being the same in all the sequences; (D) up to three bases can be inserted at any location of any of the sequences or up to three bases can be deleted from any o f the sequences; (E) all of the sequences of a said group of oligonucleotides are read 5′ to 31 or are read 3′ to 5′; and wherein each oligonucleotide of a said set has a sequence of at least ten contiguous bases of the sequence on which it is based, provided that: (F) (I) the quotient of the sum of G and C divided by the sum of A, T/U, G and C for all combined sequences of the set is between about 0.1 and 0.40 and said quotient for each sequence of the set does not vary from the quotient for the combined sequences by more than 0.2; and (II) for any phantom sequence generated from any pair of first and second sequences of the set L₁ and L₂ in length, respectively, by selection from the first and second sequences of identical bases in identical sequence with each other: (i) any consecutive sequence of bases in the phantom sequence which is identical to a consecutive sequence of bases in each of the first and second sequences from which it is generated is less than ((¾×L)−0.1) bases in length; (ii) the phantom sequence, if greater than or equal to (⅚×L) in length, contains at least three insertions/deletions or mismatches when compared to the first and second sequences from which it is generated; and (iii) the phantom sequence is not greater than or equal to ( 11/12×L) in length; where L=L₁, or if L₁≠L₂, where L is the greater of L₁ and L2; and wherein any base present may be substituted by an analogue thereof; b) mixing said cleavage means, said target nucleic acid, said first and second oligonucleotides to create a reaction mixture under reaction conditions such that at least said 5′ portion of said first oligonucleotide is annealed to said target nucleic acid and wherein at least said 5′ and central portion of said second oligonucleotide is annealed to said target nucleic acid so as to create a cleavage structure and wherein the combined melting temperature of said complementary regions within said 5′ and 3′ portions of said first oligonucleotide when annealed to said target nucleic acid is greater than the melting temperature of said 5′ and central portion of said first oligonucleotide, and wherein cleavage of said cleavage structure occurs to generate non-target cleavage products; and c) detecting said non-target cleavage products.
 6. The method of claim 5, wherein said reaction temperature is between approximately 50 and 70 degrees centigrade.
 7. The method of claim 5, wherein said target nucleic acid comprises single-stranded DNA.
 8. The method of claim 5, wherein said target nucleic acid comprises double-stranded DNA and prior to step (c), said reaction mixture is treated such that said double-stranded DNA is rendered substantially single-stranded.
 9. The method of claim 5, wherein said treatment to render said double-stranded DNA is rendered substantially single-stranded by increasing the temperature.
 10. The method of claim 5, wherein said target nucleic acid comprises RNA and wherein said first and second oligonucleotides comprise DNA.
 11. The method of claim 5, wherein said cleavage means comprises a thermostable 5′ nuclease.
 12. The method of claim 11, wherein a portion of the amino acid sequence is homologous to a portion of the amino acid sequence of a thermostable DNA polymerase derived from a thermophilic organism.
 13. The method of claim 12, wherein said organism is selected from the group consisting of Thermus aquaticus, Thermus flavus and Thermus thermophilus.
 14. The method of claim 5, wherein said source of target nucleic acid comprises a sample containing genomic DNA.
 15. The method of claim 5, wherein said reaction conditions comprise providing a source of divalent cations.
 16. The method of claim 15, wherein said divalent cation is selected from the group comprising Mn²⁺ and Mg²⁺ ions.
 17. The method of claim 5, wherein the method includes a plurality of said target nucleic acid sequences and a plurality of said second oligonucleotide molecules such that each of said second oligonucleotide molecules has a distinct 3′ region.
 18. The method of claim 5, wherein the method includes at least ten said second oligonucleotide molecules or at least eleven said second oligonucleotide molecules, or at least twelve said second oligonucleotide molecules, or at least thirteen said second oligonucleotide molecules, or at least fourteen said second oligonucleotide molecules, or at least fifteen said second oligonucleotide molecules, or at least sixteen said second oligonucleotide molecules, or at least seventeen said second oligonucleotide molecules, or at least eighteen said second oligonucleotide molecules, or at least nineteen said second oligonucleotide molecules, or at least twenty said second oligonucleotide molecules, or at least twenty-one said second oligonucleotide molecules, or at least twenty-two said second oligonucleotide molecules, or at least twenty-three said second oligonucleotide molecules, or at least twenty-four said second oligonucleotide molecules, or at least twenty-five said second oligonucleotide molecules, or at least twenty-six said second oligonucleotide molecules, or at least twenty-seven said second oligonucleotide molecules, or at least twenty-eight said second oligonucleotide molecules, or at least twenty-nine said second oligonucleotide molecules, or at least thirty said second oligonucleotide molecules, or at least thirty-one said second oligonucleotide molecules, or at least thirty-two said second oligonucleotide molecules, or at least thirty-three said second oligonucleotide molecules, or at least thirty-four said second oligonucleotide molecules, or at least thirty-five said second oligonucleotide molecules, or at least thirty-six said second oligonucleotide molecules, or at least thirty-seven said second oligonucleotide molecules, or at least thirty-eight said second oligonucleotide molecules, or at least thirty-nine said second oligonucleotide molecules, or at least forty said second oligonucleotide molecules, or at least forty-one said second oligonucleotide molecules, or at least forty-two said second oligonucleotide molecules, or at least forty-three said second oligonucleotide molecules, or at least forty-four said second oligonucleotide molecules, or at least forty-five said second oligonucleotide molecules or at least forty-six said second oligonucleotide molecules, or at least forty-seven said second oligonucleotide molecules, or at least forty-eight said second oligonucleotide molecules, or at least forty-nine said second oligonucleotide molecules, or at least fifty said second oligonucleotide molecules, or at least sixty said second oligonucleotide molecules, or at least seventy said second oligonucleotide molecules, or at least eighty said second oligonucleotide molecules, or at least ninety said second oligonucleotide molecules, or at least one hundred said second oligonucleotide molecules, or at least one hundred and ten said second oligonucleotide molecules, or at least one hundred and twenty said second oligonucleotide molecules, or at least one hundred and thirty said second oligonucleotide molecules, or at least one hundred and forty said second oligonucleotide molecules, or at least one hundred and fifty said second oligonucleotide molecules, or at least one hundred and sixty said second oligonucleotide molecules, or at least one hundred and seventy said second oligonucleotide molecules, or at least one hundred and eighty said second oligonucleotide molecules, or at least one hundred and ninety said second oligonucleotide molecules, or at least two hundred said second oligonucleotide molecules.
 19. The method of claim 5, wherein said 3′ portion of said second oligonucleotide incorporates fluorescent molecule, a radiolabelled nucleotide, digoxigenin, biotinylation and the like.
 20. A method of analyzing a biological sample comprising a plurality of target nucleic acid molecules for the presence of a mutation or polymorphism at a locus of each target nucleic acid molecule, the method comprising: a) providing: i) a cleavage means, ii) a plurality of target nucleic acid molecules, each of said target nucleic acid molecules having a first region, a second region and a third region, wherein said first region is located adjacent to and downstream from said second region, and said second region is located adjacent to and downstream from said third region; iii) a plurality of first oligonucleotide molecules, each having a 5′ and a 3′ portion, said 5′ portion of said first oligonucleotide molecules having a sequence complementary to said second region of said target nucleic acid molecules and said 3′ portion of said first oligonucleotide molecules having a sequence complementary to said third region of said target nucleic acid molecules; iv) a plurality of second oligonucleotide molecules, each having a 5′ portion, a central portion and a 3′ portion, said 5′ portion of said second oligonucleotide molecules having a sequence complementary to said first region of said target nucleic acid molecules, said central portion of said second oligonucleotide molecules having a sequence complimentary to said second region of said target nucleic acid molecules, and said 3′ portion of said second oligonucleotide molecules having a sequence that is not base-paired to said target nucleic acid and is selected from a set of oligonucleotides based on a following group of sequences, 1 4 6 6 1 3 2 4 5 5 2 3 1 8 1 2 3 4 1 7 1 9 8 4 1 1 9 2 6 9 1 2 4 3 9 6 9 8 9 8 10 9 9 1 2 3 8 10 8 8 7 4 3 1 1 1 1 1 1 2 2 1 3 3 2 2 3 1 2 2 3 2 4 1 4 4 4 2 1 2 3 3 1 1 1 3 2 2 1 4 3 3 3 3 3 4 4 3 1 1 4 4 3 4 1 1 3 3 3 6 6 6 3 5 6 6 1 1 6 5 7 6 7 7 7 5 8 7 5 5 8 8 2 1 7 7 1 1 2 3 2 3 1 3 2 6 5 6 1 6 4 8 1 1 3 8 5 3 1 1 6 3 5 6 8 8 6 6 8 3 6 5 7 3 1 2 3 1 4 6 1 5 7 5 4 3 2 1 6 7 3 6 2 6 1 3 3 1 2 7 6 8 3 1 3 4 3 1 2 5 3 5 6 1 2 7 3 6 1 7 2 7 4 6 3 5 1 7 5 4 6 3 8 6 6 8 2 3 7 1 7 1 7 8 6 3 7 3 4 1 6 8 4 7 7 1 2 4 3 6 5 2 6 3 1 4 1 4 6 1 3 3 1 4 8 1 8 3 3 5 3 8 1 3 6 6 3 7 7 3 8 6 4 7 3 1 3 7 8 6 10 9 5 5 10 10 7 10 10 10 7 9 9 9 7 7 10 9 9 3 10 3 10 3 9 6 3 4 10 6 10 4 10 3 9 4 3 9 3 10 4 9 9 10 5 9 4 8 3 9 4 9 10 7 3 5 9 4 10 8 4 10 5 4 9 3 5 3 3 9 8 10 6 8 6 9 7 10 4 6 10 9 6 4 4 9 8 10 8 3 7 7 9 10 5 3 8 8 9 3 9 10 8 10 2 9 5 9 9 6 2 2 7 10 9 7 5 3 10 6 10 3 6 8 9 2 10 9 3 2 7 3 8 9 10 3 6 2 3 2 5 10 8 9 8 2 3 10 2 9 6 3 9 8 2 10 3 7 3 9 9 10 9 10 1 1 9 4 10 1 9 1 4 1 7 1 10 9 8 1 9 1 10 1 10 6 9 6 9 1 3 10 3 10 8 8 9 1 3 8 1 9 10 3 9 10 1 3 6 9 1 9 1 10 3 1 1 4 9 6 8 10 3 3 9 6 1 10 5 3 1 6 9 10 6 1 8 10 9 6 5 9 9 4 10 3 2 10 9 1 9 5 10 10 7 2 1 9 10 9 9 1 8 2 1 8 6 8 9 10 1 9 1 3 8 10 9 6 9 10 1 2 1 10 8 9 9 2 1 9 6 7 2 9 4 3 9 3 5 1 5 11 10 14 12 1 7 12 4 13 3 2 5 5 4 4 12 9 2 13 13 11 13 13 10 2 5 4 12 7 11 7 4 11 6 4 12 12 1 9 11 11 12 9 4 14 12 6 12 7 13 2 9 11 9 11 3 4 1 3 10 5 12 11 4 4 4 13 7 12 1 5 9 13 10 11 11 6 10 14 14 10 1 3 2 14 1 10 4 5 10 12 12 7 11 10 9 11 2 12 8 11 2 8 5 2 12 14 1 8 13 3 7 8 9 4 7 5 4 2 13 2 12 7 1 12 11 10 9 7 5 11 8 12 2 2 12 7 5 2 14 3 4 13 1 8 8 1 5 9 14 5 11 10 13 3 14 1 4 13 2 4 4 4 5 11 3 10 10 9 2 3 3 11 11 4 8 14 3 4 5 1 14 8 11 2 14 3 11 6 12 5 13 4 4 1 10 1 6 10 11 6 5 1 5 8 12 5 1 7 4 5 9 6 9 2 13 2 4 4 2 3 11 2 2 5 9 3 8 1 10 12 2 8 12 7 9 11 4 1 12 1 4 14 3 13 11 2 7 10 4 1 3 4 12 11 11 11 3 3 4 2 12 11 1 5 9 4 2 1 6 1 12 2 10 5 10 5 1 12 2 14 2 11 7 9 4 11 7 4 4 5 14 12 12 5 2 1 10 12 5 9 2 11 6 1 12 14 3 6 1 14 5 9 11 10 1 4 2 5 12 14 10 10 4 5 8 4 5 6 10 12 4 6 12 5 4 2 1 13 6 8 9 10 10 14 5 3 6 14 10 11 3 3 2 9 10 12 5 7 13 3 7 10 5 12 6 4 1 2 5 13 6 1 13 4 14 13 2 12 1 14 1 9 4 11 13 2 6 10 1 10 7 4 5 8 7 2 2 10 13 4 8 2 11 4 6 14 4 8 2 6 2 3 7 1 12 11 2 9 5 6 10 4 13 4 5 10 4 11 9 3 3 11 9 3 2 3 8 15 6 20 17 19 21 10 15 3 7 11 11 7 17 20 14 9 16 6 17 13 21 21 10 15 22 6 17 21 15 7 17 10 22 22 3 20 8 15 20 16 17 21 10 16 6 22 6 21 14 14 14 16 7 17 3 20 10 7 16 19 14 17 7 21 20 16 7 15 22 10 20 10 18 11 22 18 18 7 19 15 7 22 21 18 7 21 16 3 14 13 7 22 17 13 19 7 8 12 10 17 15 3 21 14 9 7 19 6 15 7 14 14 4 17 10 15 20 19 21 6 18 4 20 16 2 19 8 17 6 13 12 12 6 17 4 20 16 21 12 10 19 16 14 14 15 2 7 21 8 16 21 6 22 16 14 17 22 14 17 20 10 21 7 15 21 18 16 13 20 18 21 12 15 7 4 22 14 13 7 19 14 8 15 4 4 5 3 20 7 16 22 18 6 18 13 20 19 6 16 3 13 3 18 6 22 7 20 18 10 17 11 21 8 13 7 10 17 19 10 14

wherein: (A) each of 1 to 22 is a 4mer selected from the group of 4mers consisting of WWWW, WWWX, WWWY, WWXW, WWXX, WWXY, WWYW, WWYX, WWYY, WXWW, WXWX, WXWY, WXXW, WXXX, WXXY, WXYW, WXYX, WXYY, WYWW, WYWX, WYWY, WYXW, WYXX, WYXY, WYYW, WYYX, WYYY, XWWW, XWWX, XWWY, XWXW, XWXX, XWXY, XWYW, XWYX, XWYY, XXWW, XXWX, XXWY, XXXW, XXXX, XXXY, XXYW, XXYX, XXYY, XYWW, XYWX, XYWY, XYXW, XYXX, XYXY, XYYW, XYYX, XYYY, YWWW, YWWX, YWWY, YWXW, YWXX, YWXY, YWYW, YWYX, YWYY, YXWW, YXWX, YXWY, YXXW, YXXX, YXXY, YXYW, YXYX, YXYY, YYWW, YYWX, YYWY, YYXW, YYXX, YYXY, YYYW, YYYX, and YYYY, and (B) each of 1 to 22 is selected so as to be different from all of the others of 1 to 22; (C) each of W, X and Y is a base in which: (i) (a) W=one of A, T/U, G, and C, X=one of A, T/U, G, and C, Y=one of A, T/U, G, and C, and each of W, X and Y is selected so as to be different from all of the others of W, X and Y,  (b) an unselected said base of (i)(a) can be substituted any number of times for any one of W, X and Y, or (ii) (a) W=G or C, X=A or T/U, Y=A or T/U, and X≠Y, and  (b) a base not selected in (ii)(a) can be inserted into each sequence at one or more locations, the location of each insertion being the same in all the sequences; (D) up to three bases can be inserted at any location of any of the sequences or up to three bases can be deleted from any of the sequences; (E) all of the sequences of a said group of oligonucleotides are read 5′ to 3′ or are read 3′ to 5′; and wherein each oligonucleotide of a said set has a sequence of at least ten contiguous bases of the sequence on which it is based, provided that: (F) (I) the quotient of the sum of G and C divided by the sum of A, T/U, G and C for all combined sequences of the set is between about 0.1 and 0.40 and said quotient for each sequence of the set does not vary from the quotient for the combined sequences by more than 0.2; and (II) for any phantom sequence generated from any pair of first and second sequences of the set L₁ and L₂ in length, respectively, by selection from the first and second sequences of identical bases in identical sequence with each other: (i) any consecutive sequence of bases in the phantom sequence which is identical to a consecutive sequence of bases in each of the first and second sequences from which it is generated is less than ((¾×L)−1) bases in length; (ii) the phantom sequence, if greater than or equal to (⅚×L) in length, contains at least three insertions/deletions or mismatches when compared to the first and second sequences from which it is generated; and (iii) the phantom sequence is not greater than or equal to ( 11/12×L) in length; where L=L₁, or if L₁≠L₂, where L is the greater of L₁ and L₂; and wherein any base present may be substituted by an analogue thereof; b) mixing said cleavage means, said target nucleic acid, said first and second oligonucleotides to create a reaction mixture under reaction conditions such that at least said 5′ portion of said first oligonucleotide is annealed to said target nucleic acid and wherein at least said 5′ and central portion of said second oligonucleotide is annealed to said target nucleic acid so as to create a cleavage structure and wherein the combined melting temperature of said complementary regions within said 5′ and 3′ portions of said first oligonucleotide when annealed to said target nucleic acid is greater than the melting temperature of said 5′ and central portion of said first oligonucleotide, and wherein cleavage of said cleavage structure occurs to generate non-target cleavage products; and c) detecting said non-target cleavage products.
 21. The method of claim 20, wherein said reaction temperature is between approximately 50 and 70 degrees centigrade.
 22. The method of claim 20, wherein said target nucleic acid molecules comprises single-stranded DNA.
 23. The method of claim 20, wherein said target nucleic acid molecules comprises double-stranded DNA and prior to step (c), said reaction mixture is treated such that said double-stranded DNA is rendered substantially single-stranded.
 24. The method of claim 20, wherein said treatment to render said double-stranded DNA is rendered substantially single-stranded by increasing the temperature.
 25. The method of claim 20, wherein said target nucleic acid molecules comprises RNA and wherein said first and second oligonucleotide molecules comprise DNA.
 26. The method of claim 20, wherein said cleavage means comprises a thermostable 5′ nuclease.
 27. The method of claim 26, wherein a portion of the amino acid sequence is homologous to a portion of the amino acid sequence of a thermostable DNA polymerase derived from a thermophilic organism.
 28. The method of claim 28, wherein said organism is selected from the group consisting of Thermus aquaticus, Thermus flavus and Thermus thermophilus.
 29. The method of claim 20, wherein said source of target nucleic acid molecules comprises a sample containing genomic DNA.
 30. The method of claim 20, wherein said reaction conditions comprise providing a source of divalent cations.
 31. The method of claim 30, wherein said divalent cation is selected from the group comprising Mn²⁺ and Mg²⁺ ions.
 32. The method of claim 20, wherein the method includes a plurality of said target nucleic acid sequences and a plurality of said second oligonucleotide molecules such that each of said second oligonucleotide molecules has a distinct 3′ region.
 33. The method of claim 20, wherein the method includes at least ten said second oligonucleotide molecules or at least eleven said second oligonucleotide molecules, or at least twelve said second oligonucleotide molecules, or at least thirteen said second oligonucleotide molecules, or at least fourteen said second oligonucleotide molecules, or at least fifteen said second oligonucleotide molecules, or at least sixteen said second oligonucleotide molecules, or at least seventeen said second oligonucleotide molecules, or at least eighteen said second oligonucleotide molecules, or at least nineteen said second oligonucleotide molecules, or at least twenty said second oligonucleotide molecules, or at least twenty-one said second oligonucleotide molecules, or at least twenty-two said second oligonucleotide molecules, or at least twenty-three said second oligonucleotide molecules, or at least twenty-four said second oligonucleotide molecules, or at least twenty-five said second oligonucleotide molecules, or at least twenty-six said second oligonucleotide molecules, or at least twenty-seven said second, oligonucleotide molecules, or at least twenty-eight said second oligonucleotide molecules, or at least twenty-nine said second oligonucleotide molecules, or at least thirty said second oligonucleotide molecules, or at least thirty-one said second oligonucleotide molecules, or at least thirty-two said second oligonucleotide molecules, or at least thirty-three said second oligonucleotide molecules, or at least thirty-four said second oligonucleotide molecules, or at least thirty-five said second oligonucleotide molecules, or at least thirty-six said second oligonucleotide molecules, or at least thirty-seven said second oligonucleotide molecules, or at least thirty-eight said second oligonucleotide molecules, or at least thirty-nine said second oligonucleotide molecules, or at least forty said second oligonucleotide molecules, or at least forty-one said second oligonucleotide molecules, or at least forty-two said second oligonucleotide molecules, or at least forty-three said second oligonucleotide molecules, or at least forty-four said second oligonucleotide molecules, or at least forty-five said second oligonucleotide molecules, or at least forty-six said second oligonucleotide molecules, or at least forty-seven said second oligonucleotide molecules, or at least forty-eight said second oligonucleotide molecules, or at least forty-nine said second oligonucleotide molecules, or at least fifty said second oligonucleotide molecules, or at least sixty said second oligonucleotide molecules, or at least seventy said second oligonucleotide molecules, or at least eighty said second oligonucleotide molecules, or at least ninety said second oligonucleotide molecules, or at least one hundred said second oligonucleotide molecules, or at least one hundred and ten said second oligonucleotide molecules, or at least one hundred and twenty said second oligonucleotide molecules, or at least one hundred and thirty said second oligonucleotide molecules, or at least one hundred and forty said second oligonucleotide molecules, or at least one hundred and fifty said second oligonucleotide molecules, or at least one hundred and sixty said second oligonucleotide molecules, or at least one hundred and seventy said second oligonucleotide molecules, or at least one hundred and eighty said second oligonucleotide molecules, or at least one hundred and ninety said second oligonucleotide molecules, or at least two hundred said second oligonucleotide molecules.
 34. The method of claim 20, wherein said 3′ portion of said second oligonucleotide molecules incorporates a fluorescent molecule, a radiolabelled nucleotide, digoxigenin, biotinylation and the like.
 35. A composition comprising a cleavage structure, said cleavage structure comprising: a) a target nucleic acid having a first region, a second region and a third region, wherein said first region is located adjacent to and downstream from said second region and said second region is located to and downstream from said third region; b) a first oligonucleotide having a 5′ portion and a 3′ portion, said 5′ portion of said first oligonucleotide having a sequence complementary to said second region of said target nucleic acid and said 3′ portion of said first oligonucleotide having a sequence complementary to said third region of said target nucleic acid; and c) a second oligonucleotide having a 5′ portion, a central portion and a 3′ portion, said 5′ portion of said second oligonucleotide having a sequence complementary to said first region of said target nucleic acid, said central region of said second oligonucleotide having a sequence complementary to said second region of said target nucleic acid, and said 3′ portion of said second oligonucleotide having a sequence that is not base-paired to said target nucleic acid and is selected from a set of oligonucleotides based on a following group of sequences: 1 1 1 2 2 3 2 3 1 1 1 3 1 2 2 3 2 2 2 3 2 3 2 1 3 2 2 1 3 1 3 2 2 1 1 2 2 3 2 1 2 2 2 3 1 2 3 1 1 2 3 2 2 1 1 1 3 2 1 1 3 2 3 2 2 3 1 1 1 2 3 2 2 3 1 2 3 2 2 1 3 1 1 3 2 1 2 1 2 2 3 2 3 1 1 2 2 2 2 3 2 3 2 1 3 1 1 2 1 2 3 2 3 2 2 3 2 2 1 1 1 2 1 1 3 2 3 2 1 1 3 2 3 1 1 1 2 1 1 3 1 1 3 1 1 1 3 1 3 2 1 2 2 2 3 2 2 3 2 3 1 3 2 2 1 1 1 2 3 2 3 2 2 2 1 2 3 2 2 1 2 1 2 3 2 3 1 1 3 2 2 2 1 1 1 3 1 3 1 1 2 1 3 1 1 2 1 2 3 2 3 2 1 1 3 2 2 1 2 3 1 1 1 3 1 3 2 3 1 3 1 2 1 1 2 3 2 2 2 1 1 2 3 1 3 1 1 1 2 1 2 3 2 2 1 3 1 1 2 3 2 3 1 2 2 2 1 3 2 2 3 2 2 3 1 2 3 2 2 2 1 3 2 1 3 2 2 2 3 2 1 1 1 3 1 3 2 1 2 1 1 3 2 2 2 3 1 2 3 1 2 1 1 1 1 3 2 1 1 3 1 1 2 3 1 2 3 2 1 1 2 1 1 3 2 3 3 2 1 3 1 1 1 2 1 3 2 2 2 1 2 2 3 1 2 3 1 2 2 3 2 3 2 1 1 3 2 3 1 1 1 2 1 3 2 3 1 3 2 2 1 2 2 2 1 1 1 2 1 3 1 2 3 1 2 1 2 1 1 3 2 3 1 3 1 1 2 3 1 2 1 1 3 2 2 1 2 1 1 3 2 3 2 2 1 2 3 2 3 1 3 2 2 1 2 1 3 1 2 1 1 1 3 1 3 1 2 3 1 2 2 2 3 2 2 3 1 3 1 3 2 2 3 1 3 1 1 2 3 2 1 2 1 3 2 1 2 2 1 2 1 1 3 2 1 3 2 2 2 3 2 1 1 3 1 1 2 3 1 2 2 3 2 1 2 2 1 2 3 1 1 1 2 2 3 1 3 2 3 1 1 3 1 2 2 3 1 2 3 2 1 2 1 2 3 2 1 1 1 2 2 3 2 2 1 2 3 2 2 3 1 3 3 1 1 2 2 3 2 1 2 1 1 1 3 2 1 2 2 1 3 1 2 3 2 3 2 1 3 1 2 3 1 3 1 2 2 1 1 3 2 3 2 2 1 2 2 2 3 1 3 2 2 1 1 3 2 2 2 3 2 2 2 1 2 3 2 1 2 1 3 1 1 3 3 1 3 2 1 2 2 1 3 2 1 1 1 3 2 3 1 2 1 2 3 1 2 1 3 2 3 1 1 2 3 1 2 2 2 1 3 2 1 1 1 2 3 1 2 2 3 1 3 1 2 2 3 1 1 3 2 2 1 2 1 3 1 1 1 2 3 1 2 2 1 3 1 3 2 3 1 2 1 1 1 2 3 2 2 1 3 2 2 3 1 1 2 2 3 2 2 1 2 1 2 1 3 2 1 1 1 2 3 2 2 2 3 2 3 2 3 2 2 3 2 2 1 1 3 2 3 2 2 1 3 2 2 1 2 2 2 3 2 2 3 2 1 3 3 2 1 3 2 1 1 2 1 2 3 1 1 3 2 3 1 3 1 1 2 1 2 1 2 1 3 2 3 2 1 2 1 3 1 1 2 3 2 1 3 1 2 2 2 1 3 2 2 2 3 2 1 3 1 2 2 1 3 1 2 3 2 3 2 2 2 3 2 1 1 1 2 1 3 2 1 2 1 3 1 3 2 1 3 1 3 1 2 3 1 2 1 2 2 2 1 2 2 3 2 3 1 1 1 3 1 1 1 3 1 3 1 1 3 1 1 1 2 2 2 3 2 3 1 3 1 1 2 2 1 1 3 1 2 2 1 1 3 1 1 2 3 2 1 2 1 2 2 1 3 2 2 1 1 3 1 1 1 3 1 1 3 1 3 2 2 3 2 2 3 2 1 3 2 2 3 1 3 1 1 1 2 1 2 3 2 1 3 2 2 2 2 1 3 1 3 2 2 3 2 2 1 1 1 3 1 3 2 3 2 1 1 1 2 1 3 2 2 1 2 3 1 2 3 2 3 2 1 2 1 1 3 2 1 1 2 1 2 3 2 2 2 3 2 2 1 3 1 1 2 3 1 3 1 1 3 1 2 2 2 1 2 3 1 3 2 1 2 1 3 2 2 2 1 1 1 3 1 1 3 2 1 3 2 1 3 1 3 2 3 1 3 1 2 1 2 1 3 1 2 2 2 1 3 1 1 1 3 2 1 1 2 2 3 2 2 2 1 2 1 3 2 3 1 1 3 2 3 1 1 2 1 3 2 1 1 1 3 2 1 1 3 2 1 3 2 1 1 2 1 3 2 3 2 3 2 2 1 1 1 2 2 2 3 2 3 1 3 2 2 1 2 3 1 1 1 3 1 2 1 1 3 1 3 1 1 1 3 2 1 3 1 3 1 1 2 1 1 1 3 1 2 1 1 3 1 1 1 2 2 2 1 1 3 1 2 2 3 2 2 1 1 3 1 3 2 1 3 1 1 3 3 2 2 2 1 1 1 3 1 2 2 3 2 1 1 3 1 1 2 3 2 3 2 1 2 2 2 3 2 3 1 1 3 1 2 3 1 1 3 2 1 2 2 2 3 2 1 2 2 3 2 3 2 2 2 1 3 1 1 2 2 2 1 3 2 1 2 3 2 3 2 1 3 1 2 1 1 2 3 1 2 2 1 2 1 3 1 1 1 3 2 3 2 2 2 3 3 2 2 1 2 2 2 3 2 1 1 3 2 2 1 1 3 1 2 1 3 2 1 3 1 3 2 2 2 1 2 2 3 1 1 1 3 1 3 2 2 2 3 1 1 2 1 3 2 2 3 2 3 2 2 2 1 2 2 3 2 3 2 1 3 2 2 2 1 1 1 3 1 2 2 3 2 3 1 3 1 1 3 1 2 1 2 3 1 1 1 3 2 2 1 2 2 3 1 3 1 1 2 3 2 1 1 1 3 1 1 2 3 2 2 2 1 2 2 3 1 2 3 2 3 1 1 1 3 2 2 1 2 3 1 2 3 2 2 1 1 2 2 3 3 2 2 2 1 3 2 1 2 2 1 3 2 2 3 2 2 1 1 3 1 2 2 3 3 1 2 2 3 1 2 1 2 2 2 3 1 1 2 3 2 2 2 3 2 2 2 3 2 3 1 1 2 2 3 1 1 1 3 2 3 2 1 1 2 3 2 2 3 2 1 2 3 1 2 2 3 2 1 2 2 3 2 2 3 1 3 1 1 2 1 3 1 1 2 1 1 1 1 2 2 2 3 1 3 1 2 2 2 3 2 3 1 2 1 3 1 3 2 1 3 2 1 1 2 2 1 3 1 2 2 2 3 2 2 2 3 2 2 3 2 2 3 2 3 2 2 2 3 2 1 2 2 3 2 2 1 3 2 3 1 1 2 1 2 1 3 2 1 2 3 2 1 3 2 1 3 2 1 3 1 2 3 2 2 2 1 2 3 1 1 2 2 3 2 2 2 1 1 1 3 1 2 3 1 2 2 3 1 1 3 1 1 1 2 3 2 3 2 3 1 2 1 1 2 3 1 2 3 2 2 1 2 2 2 3 2 3 2 1 1 2 1 3 2 2 3 2 3 1 3 1 1 2 2 2 3 2 1 1 2 2 1 3 1 2 1 3 1 2 3 2 1 1 3 1 3 1 1 1 2 2 3 2 3 1 1 1 1 3 1 2 2 1 1 3 1 3 1 1 3 2 2 1 1 2 1 3 1 3 2 1 3 1 1 3 2 1 1 1 2 2 3 2 3 1 1 2 3 1 1 1 3 1 1 1 1 1 2 3 2 1 1 3 1 1 1 3 1 1 3 1 2 2 3 2 2 3 2 1 2 2 2 3 1 2 2 2 1 2 3 2 3 2 2 1 2 3 2 2 3 1 3 2 3 2 1 2 2 3 1 3 1 1 1 2 2 2 3 1 1 3 1 1 2 3 1 1 3 1 1 2 2 3 2 1 2 3 1 1 1 2 3 1 1 2 2 3 2 1 1 3 2 1 2 2 3 2 1 3 1 1 3 2 1 1 1 3 2 2 1 3 1 1 3 2 2 2 2 1 2 3 2 1 1 2 3 1 2 1 1 3 2 3 2 1 3 2 2 3 1 2 1 2 1 3 2 2 3 1 1 1 2 2 3 2 3 1 2 1 3 2 3 2 1 2 1 1 3 1 1 1 2 2 1 3 1 3 1 3 2 2 3 2 1 1 1 3 3 1 1 2 2 3 2 3 1 1 1 2 3 2 3 1 2 2 3 1 2 1 2 1 1 1 1 2 1 1 3 2 1 3 2 2 2 1 1 2 3 1 3 1 3 1 1 3 3 1 2 2 1 1 1 3 1 1 3 2 1 1 3 2 3 1 1 2 3 2 2 2 2 1 2 3 2 3 2 3 2 2 3 2 2 2 1 3 2 3 2 2 1 2 2 1 3 1 3 2 2 1 2 1 2 3 2 1 3 2 2 1 3 1 3 2 2 1 2 1 3 1 1 1 3 1 1 1 3 1 1 3 2 3 2 2 1 1 3 2 2 1 1 1 2 1 3 2 1 2 2 1 3 2 1 1 3 2 1 2 3 2 3 1 2 2 3 2 2 2 3 2 3 2 3 1 2 2 3 1 1 2 1 2 2 3 2 3 1 1 1 2 1 2 3 2 3 1 1 1 3 1 3 2 2 1 1 3 2 3 1 2 2 1 1 1 3 1 2 2 3 1 1 2 3 1 2 2 3 1 3 1 2 1 2 3 2 1 1 1 1 1 3 1 2 3 1 2 1 3 2 2 1 1 3 2 3 2 1 1 3 2 2 1 2 1 3 2 2 3 2 2 1 2 2 3 1 3 1 1 2 2 2 1 3 1 1 3 2 2 2 1 2 1 3 2 3 1 1 2 2 1 2 3 1 3 2 3 1 1 1 3 3 1 2 1 3 1 2 2 2 1 3 1 1 2 3 1 1 2 2 1 1 3 2 3 2 2 2 3 1 1 3 1 1 3 1 3 1 2 2 2 3 1 1 1 2 2 3 1 1 2 3 1 1 2 1 1 3 1 3 2 2 3 1 2 1 1 1 2 3 2 3 1 2 3 2 2 2 1 2 3 2 1 3 2 3 2 1 3 1 2 2 3 1 1 2 2 2 2 2 1 1 3 2 3 1 3 2 2 1 2 1 3 1 1 3 2 1 3 2 1 3 1 2 2 2 1 2 3 2 3 2 2 2 3 1 1 3 2 2 1 1 3 1 2 2 1 3 2 2 1 3 1 3 1 1 1 3 2 3 1 2 1 1 1 3 2 2 1 3 2 1 1 2 3 1 2 1 1 2 3 1 1 3 2 3 2 1 2 1 2 1 3 1 1 2 3 1 1 3 2 3 2 2 1 3 2 1 2 1 3 1 2 1 3 2 1 2 1 1 1 2 2 3 1 3 2 2 2 3 2 2 2 3 1 2 2 3 2 1 3 2 1 1 2 3 1 1 3 1 1 2 1 1 3 2 1 2 3 1 3 2 3 2 2 1 1 1 2 3 2 1 1 2 1 3 2 3 2 2 3 2 2 1 3 2 2 1 3 1 3 1 3 2 2 1 3 2 3 1 1 1 2 3 2 2 3 2 2 1 1 1 2 3 1 1 1 2 1 3 1 1 1 2 3 2 1 2 2 3 2 2 2 3 2 3 1 1 3 2 2 1 2 1 1 3 2 2 2 3 2 3 1 3 1 1 2 2 1 1 3 3 1 3 2 2 2 1 2 1 3 2 2 1 3 1 1 2 1 2 3 2 2 3 2 1 3 1 3 2 2 1 2 2 1 3 1 1 3 1 1 3 1 2 2 2 1 1 3 3 1 3 2 2 1 1 2 3 1 1 1 2 1 1 3 2 1 2 2 2 3 2 3 1 2 3 1 2 3 1 1 2 1 3 2 2 3 1 1 3 2 1 2 1 2 1 3 1 2 1 3 1 2 1 2 3 1 3 1 2 3 1 1 1 3 2 2 1 3 2 1 2 1 2 3 2 1 1 1 3 1 1 1 3 2 3 1 1 1 3 1 1 3 1 1 2 3 1 1 2 3 2 1 3 1 1 1 2 3 1 1 2 3 2 2 3 1 1 1 1 1 2 2 3 1 1 2 1 3 2 3 2 3 2 3 1 3 2 2 2 1 1 2 1 3 1 2 1 2 2 3 2 2 2 3 1 2 2 1 1 2 3 1 1 3 1 3 1 1 1 3 2 2 3 2 1 1 1 3 2 2 3 1 1 3 1 2 1 1 1 3 3 2 2 1 1 3 1 3 1 2 2 1 2 3 1 3 1 2 3 2 1 2 2 1 1 3 1 1 3 1 2 1 2 1 1 3 1 1 3 1 2 2 3 1 1 2 2 3 3 2 1 3 1 1 1 2 2 2 3 1 1 2 2 3 1 2 3 2 3 1 1 1 1 1 3 1 3 2 1 3 1 2 2 3 1 2 1 1 3 2 1 2 1 2 3 1 2 3 1 2 1 2 1 3 2 1 3 2 3 1 1 3 1 1 1 2 1 1 3 2 1 3 1 2 1 1 2 3 1 2 3 1 3 1 1 1 2 3 1 1 3 1 2 1 1 2 3 2 3 1 1 1 3 2 1 2 2 2 3 2 3 1 2 1 2 1 3 2 1 1 2 1 1 3 1 3 1 1 2 2 3 1 2 1 2 3 1 1 3 1 2 3 2 1 1 3 2 3 2 1 2 2 2 1 3 2 1 3 1 1 2 3 1 1 3 2 2 1 2 3 2 2 1 3 1 2 2 2 3 2 2 3 1 3 1 2 2 3 1 2 1 3 2 2 2 3 2 1 2 3 1 1 3 1 3 1 2 1 3 2 1 2 2 2 3 1 3 1 1 1 2 3 2 2 1 2 3 2 1 2 2 2 1 3 2 1 3 2 2 1 2 3 2 3 1 3 1 1 2 3 2 3 2 2 2 3 1 2 2 2 1 1 3 2 1 2 3 2 2 2 3 2 2 2 1 2 1 3 1 1 2 3 2 1 2 3 3 1 3 2 1 2 1 2 1 3 1 1 3 1 1 1 3 1 1 1 2 2 2 3 1 2 3 1 3 2 3 1 1 3 2 1 1 1 2 3 2 1 3 2 2 1 2 2 2 2 1 1 3 1 1 3 2 3 1 3 2 2 1 2 2 3 2 3 1 2 1 2 1 2 3 1 1 1 2 3 1 3 1 1 2 1 2 2 3 2 2 3 2 2 2 3 3 1 2 2 1 1 2 3 1 2 2 1 2 3 2 3 1 1 2 2 3 1 2 3 3 1 1 1 2 3 2 2 1 1 1 3 1 2 1 2 3 1 1 1 3 2 1 3 2 1 2 2 3 2 2 3 1 2 2 2 3 1 2 1 2 2 1 3 2 3 2 3 2 2 2 1 2 3 2 2 2 3 2 3 2 1 2 3 2 1 1 3 2 1 3 2 1 1 2 2 3 1 1 1 3 1 1 2 2 3 2 3 2 3 1 1 2 2 3 1 2 3 1 3 2 2 2 3 1 1 2 2 2 3 2 2 2 3 1 3 2 1 1 2 3 1 2 3 2 1 2 1 1 2 3 1 2 3 2 3 2 3 2 1 1 1 2 2 1 2 3 2 3 1 3 1 3 1 1 3 1 1 2 2 2 3 2 2 2 1 2 2 3 2 3 1 2 1 1 1 3 2 1 2 2 3 2 2 3 1 2 1 3 1 1 1 3 1 1 3 2 1 3 1 1 2 1 3 1 1 1 3 2 2 1 1 2 1 3 1 2 2 3 2 3 2 1 3 2 2 1 1 3 1 3 2 2 3 2 2 2 1 1 2 2 1 3 2 1 3 2 1 1 3 2 2 3 2 2 1 3 1 1 2 1 3 2 2 1 1 2 2 2 3 1 1 3 2 1 2 1 1 2 3 1 1 2 3 2 3 2 3 2 1 3 1 1 1 2 2 3 2 1 3 2 1 2 2 2 3 1 3 1 3 1 1 2 3 2 1 2 1 2 3 2 2 1 1 2 3 1 3 1 2 3 2 2 3 2 1 2 1 2 2 2 3 1 2 1 1 3 1 3 1 1 2 3 1 1 3 1 1 3 2 2 2 3 1 1 2 1 3 2 3 2 1 1 2 3 1 1 2 1 2 3 1 2 3 3 2 1 3 2 2 2 3 2 3 1 1 2 1 3 1 1 2 2 1 3 2 2 2 1 1 1 3 1 2 3 1 2 2 3 2 1 1 2 2 2 3 2 3 2 3 1 1 3 1 1 3 1 2 2 3 2 2 3 1 3 2 2 1 1 2 1 3 1 2 1 1 1 3 1 2 2 1 2 3 2 1 3 2 3 1 2 3 2 1 1 1 2 3 2 2 3 1 1 2 2 2 1 3 1 2 3 2 1 3 1 2 1 2 3 1 1 2 3 2 3 1 2 1 3 1 1 3 2 3 2 1 2 2 1 1 3 2 1 1 3 2 2 1 2 1 2 3 1 1 2 2 1 2 3 1 3 1 1 3 1 1 2 1 3 1 3 2 2 2 2 3 2 2 1 2 3 1 1 3 2 3 1 2 2 2 3 2 2 2 3 2 3 2 1 1 1 3 1 2 2 3 2 3 2 2 1 2 1 2 3 1 1 1 2 3 2 2 3 2 3 1 2 1 3 2 1 3 2 2 1 3 1 2 1 2 2 2 3 2 3 1 1 2 2 1 1 3 1 2 1 1 1 3 1 1 3 1 3 1 1 3 2 1 3 1 2 2 3 2 1 3 1 1 2 3 1 1 2 2 2 3 2 1 3 2 1 2 1 1 1 2 1 1 3 1 3 1 3 1 3 1 1 2 3 1 2 2 2 1 3 2 1 1 2 2 1 2 3 2 3 1 1 2 1 3 1 2 2 3 2 2 3 1 1 3 2 2 1 1 3 1 2 2 2 1 2 3 2 3 1 2 1 3 2 1 3 1 3 2 2 2 1 1 1 3 1 2 1 3 2 3 2 2 2 3 2 2 3 2 3 2 2 1 2 1 2 2 3 1 2 2 2 1 2 3 1 1 3 1 3 2 1 2 1 3 2 3 1 1 1 2 2 2 3 1 2 3 1 3 2 1 3 2 2 2 1 1 3 1 3 1 1 2 1 1 1 3 2 2 3 2 2 2 3 1 2 3 2 2 2 3 1 1 2 3 3 1 2 2 3 2 3 1 2 3 1 1 2 1 1 2 3 2 2 1 2 2 3 1 3 1 2 3 1 1 3 1 1 1 2 1 2 3 1 2 1 2 3 1 1 2 1 3 2 2 1 1 1 3 2 2 1 2 2 3 1 1 3 2 3 1 1 3 2 2 3 1 2 2 3 2 1 1 3 1 1 1 2 1 3 1 3 1 2 2 2 3 2 3 2 2 3 1 3 1 2 2 3 1 3 2 2 2 1 1 3 2 1 2 2 1 3 1 2 2 1 3 2 3 1 2 1 1 2 1 3 1 1 2 3 1 2 1 1 1 2 3 2 3 3 1 2 1 1 2 1 3 2 3 1 1 2 2 2 3 1 3 2 2 3 2 1 2 1 3 1 2 1 2 2 2 3 2 1 3 2 1 3 1 1 1 3 2 1 2 3 2 3 2 2 1 2 3 1 1 2 3 2 2 3 1 1 2 2 2 3 1 1 2 3 2 1 2 3 1 1 1 3 1 2 2 2 1 3 2 2 3 2 3 1 3 1 2 1 2 1 1 1 2 1 3 1 3 1 1 3 2 2 1 2 3 1 2 3 2 3 1 2 1 2 2 1 3 2 3 1 3 1 1 1 2 3 2 2 2 1 1 2 3 2 3 1 2 2 3 1 1 3 1 1 2 1 2 3 2 3 1 1 1 2 2 1 3 2 2 2 3 3 2 2 2 3 1 2 1 3 2 2 2 1 1 2 3 1 3 2 1 2 2 3 1 3 2 2 3 2 1 1 3 2 1 1 2 3 1 2 1 1 1 3 2 1 2 3 1 2 1 1 3 1 3 2 1 3 2 1 1 2 2 3 2 2 3 2 2 2 1 3 1 2 2 2 3 1 3 1 3 1 3 2 1 2 3 2 1 2 3 1 2 2 1 2 2 1 2 2 3 1 2 2 3 2 3 1 1 2 2 1 3 1 2 1 3 1 1 3 1 3 1 2 2 1 3 2 1 2 2 2 1 3 2 1 3 2 1 1 2 1 3 1 3 2 1 2 3 2 1 2 2 1 3 1 3 1 2 1 2 2 3 1 1 1 3 2 3 2 1 2 3 2 3 1 1 1 3 2 1 1 2 3 1 2 1 1 1 2 3 1 3 3 2 1 1 2 2 1 3 2 1 1 2 3 1 2 2 2 3 1 1 2 3 1 3 3 2 2 2 1 2 2 3 2 1 1 1 3 1 2 3 2 1 1 2 3 3 1 1 2 1 3 2 1 3 1 1 2 2 3 2 2 3 2 2 1 1 1 3 1 1 2 3 2 1 2 2 3 2 2 1 3 2 2 1 2 3 2 1 3 2 3 2 3 2 1 1 3 1 3 2 3 1 1 1 3 2 2 1 2 1 2 3 1 1 1 3 2 1 2 1 1 2 1 2 1 3 1 1 3 2 2 3 1 2 3 1 3 2 2 2 1 2 3 1 2 2 2 1 3 1 1 3 2 1 1 3 1 1 2 1 1 3 2 3 1 3 2 1 2 3 2 3 2 1 2 1 1 3 2 1 1 2 2 2 3 2 1 1 3 1 1 3 2 1 3 1 1 3 1 3 2 2 3 2 1 2 2 3 2 2 1 2 1 1 3 2 3 2 3 2 2 1 2 2 1 3 2 2 3 1 1 3 2 2 1 3 1 3 2 1 1 1 2 1 2 1 3 2 3 1 2 3 2 3 1 1 1 2 2 3 1 1 2 3 2 2 1 3 1 3 1 1 2 1 3 1 3 2 3 1 2 2 1 2 1 3 2 2 3 1 1 3 2 3 1 3 2 2 1 1 2 3 1 2 2 2 3 2 1 1 1 2 1 1 2 3 2 1 1 1 3 2 1 1 1 3 1 1 1 3 2 3 1 2 3 1 3 2 2 1 3 2 2 1 2 3 1 2 3 1 1 2 1 2 2 3 2 3 2 1 1 1 1 2 3 1 3 2 2 1 3 1 3 2 1 3 1 1 2 2 1 2 3 2 3 1 2 1 2 1 3 1 1 3 1 2 2 1 3 2 2 1 3 2 3 1 2 1 1 2 1 3 2 2 2 3 2 2 3 1 3 1 2 2 2 1 2 3 1 3 2 1 2 1 3 1 1 2 1 3 2 2 1 3 2 1 3 2 1 1 3 1 3 2 1 2 3 1 1 2 2 2 3 2 1 2 2 3 2 3 1 1 3 2 2 2 1 3 2 1 3 2 1 3 2 1 1 3 1 1 3 1 3 1 1 2 2 1 3 1 2 2 1 1 1 1 2 3 2 3 2 2 1 2 3 2 1 2 3 2 1 1 1 2 1 3 2 3 3 1 1 2 2 1 3 2 2 1 3 1 3 2 1 1 1 2 2 3 2 2 2 3 3 1 1 1 2 2 3 1 1 3 1 2 1 3 2 1 1 3 1 1 1 2 3 1 3 2 3 2 1 2 2 1 2 3 2 3 1 2 2 2 1 2 3 1 2 1 3 1 2 1 2 2 1 2 3 1 3 1 1 1 3 2 2 3 1 1 2 1 3 2 1 3 2 1 2 3 2 1 2 2 3 2 1 2 2 3 1 3 2 1 3 1 2 3 1 1 3 2 3 1 2 2 3 1 1 2 1 3 2 1 3 1 2 2 3 2 2 2 1 1 1 3 2 1 1 3 2 2 3 2 2 2 3 1 2 2 3 1 1 1 2 2 2 3 3 1 1 3 2 2 2 3 1 2 2 2 1 1 3 2 2 2 1 1 3 1 1 3 3 1 3 1 1 3 1 2 1 1 1 2 3 1 2 1 2 2 3 2 2 1 2 3 1 2 3 1 2 3 1 3 2 2 3 2 2 1 1 2 1 3 2 2 1 3 2 2 2 1 2 3 1 2 1 2 2 2 3 1 1 3 1 3 2 3 2 2 1 1 3 1 3 1 3 1 2 3 1 2 2 1 1 1 3 2 3 1 2 2 2 1 2 3 1 1 1 2 1 3 2 2 1 1 3 1 3 2 3 1 2 3 1 3 1 1 2 1 1 1 2 2 2 1 2 2 3 2 2 1 3 1 2 1 1 1 3 1 3 2 2 3 1 3 1 3 1 1 2 1 2 2 3 1 2 1 3 2 2 3 1 1 3 2 2 3 1 1 2 1 3 2 3 2 1 1 1 3 2 3 2 1 3 1 2 2 3 2 1 1 1 2 1 3 2 1 3 2 3 1 2 1 2 3 1 2 2 2 3 1 1 2 1 2 2 3 2 3 2 1 2 2 3 1 1 2 2 1 3 1 1 2 1 3 2 3 1 3 1 1 2 3 1 2 1 2 3 1 3 1 2 1 3 1 1 3 2 2 2 1 1 2 3 2 3 1 1 3 1 1 3 2 1 1 3 2 1 2 1 1 1 3 2 1 1 1 2 3 2 2 2 1 1 3 2 3 2 3 1 2 1 1 3 1 1 1 3 1 2 1 3 1 2 1 2 2 3 2 2 3 1 1 2 3 2 3 2 2 2 1 1 1 3 1 3 1 3 1 1 2 1 1 2 3 1 2 3 1 3 1 2 3 1 2 2 1 2 2 3 1 2 1 3 1 3 1 1 1 3 1 3 1 3 1 1 2 2 3 2 1 2 2 1 1 1 2 3 2 1 2 1 1 2 3 1 3 1 2 1 2 3 2 2 2 3 2 3 1 1 1 2 1 3 1 2 1 1 3 1 2 2 3 1 2 2 3 2 3 2 2 2 3 2 2 2 3 1 2 3 1 2 1 1 2 1 3 1 1 3 1 3 1 1 2 3 1 1 3 1 2 3 1 1 2 1 1 3 2 2 3 2 3 1 1 2 3 2 2 2 1 1 3 1 2 3 1 1 1 3 1 1 1 3 2 3 2 1 3 1 1 2 1 2 2 2 3 2 2 1 1 1 2 3 2 1 2 3 2 1 3 2 1 1 2 2 3 1 3 2 1 3 2 1 3 2 3 2 3 1 1 3 2 2 1 2 2 2 3 2 2 1 2 1 3 2 3 1 1 2 3 2 2 2 3 2 1 1 1 3 1 3 2 2 2 1 1 3 1 2 1 1 1 2 3 1 3 1 1 2 2 3 1 3 2 1 1 2 2 3 2 2 3 1 2 3 1 3 1 1 1 2 2 3 2 2 2 1 1 3 2 3 2 2 2 1 1 1 2 1 1 3 2 1 3 2 3 2 3 1 3 2 1 1 2 1 3 2 1 2 1 2 3 1 1 1 2 1 2 3 2 3 1 2 1 3 2 1 1 3 1 3 1 1 2 2 3 2 1 1 3 1 3 2 3 1 2 2 1 2 1 3 1 2 3 1 2 1 3 1 3 2 1 1 3 1 1 2 3 1 1 1 3 1 3 1 2 1 1 2 1 2 1 1 3 2 1 1 3 2 1 3 1 2 3 2 2 1 1 1 3 1 3 1 2 1 1 1 2 1 3 1 1 1 3 1 1 2 2 3 2 1 3 1 3 2 1 3 2 1 2 1 3 1 2 2 2 1 1 3 2 3 1 1 3 1 3 1 3 2 2 1 2 3 1 1 2 3 2 2 2 3 2 1 1 1 2 3 2 1 2 1 3 1 2 1 3 1 1 1 2 1 3 1 1 2 3 1 3 2 1 3 2 3 1 1 1 2 1 2 3 2 2 3 1 1 2 2 1 2 3 2 1 3 1 3 1 1 1 3 2 1 1 1 3 2 1 3 2 1 1 1 2 2 3 1 3 1 3 2 1 3 2 2 3 1 1 2 2 2 3 2 1 1 1 3 2 3 2 2 2 1 2 1 3 2 3 2 3 2 1 1 2 1 2 1 2 3 1 2 2 2 3 1 3 1 2 3 1 3 1 1 2 3 2 1 1 1 1 2 1 2 2 3 1 2 1 2 3 2 3 2 2 3 2 3 1 1 3 2 1 1 3 2 3 1 3 1 2 2 1 2 3 1 3 2 1 2 2 3 1 2 2 2 1 2 2 3 2 1 2 2 2 1 3 1 2 1 3 2 3 1 3 1 2 2 1 2 3 1 2 1 3 1 1 1 2 3 1 1 1 3 1 2 1 3 1 2 1 3 1 1 3 3 1 2 2 3 2 1 2 1 2 3 2 1 1 1 3 2 1 3 2 2 2 1 3 2 1 2 3 1 1 2 3 2 2 1 2 2 3 2 3 2 3 2 2 3 1 2 2 3 1 2 1 2 2 1 3 2 1 3 1 3 2 1 1 3 2 1 2 1 2 2 3 2 3 1 3 1 2 3 1 1 2 2 2 3 2 3 2 2 1 2 3 1 2 1 2 2 1 2 3 1 1 2 3 1 1 3 2 1 1 1 3 1 3 1 2 3 2 1 1 3 1 3 2 3 1 1 2 2 2 3 2 2 3 2 1 1 2 2 2 3 2 2 2 1 3 1 1 1 2 2 3 2 1 3 1 3 2 2 1 1 2 2 3 2 3 2 1 3 2 3 2 2 1 1 2 3 1 1 1 3 2 2 3 2 3 1 1 2 1 1 2 2 3 2 3 1 2 2 2 3 2 2 1 1 3 1 1 3 1 2 2 1 1 2 3 1 3 2 1 3 2 1 2 2 3 2 1 1 1 3 2 1 2 1 1 1 3 1 3 2 3 1 2 2 3 2 2 3 2 1 2 1 3 2 2 1 2 2 3 2 3 2 1 3 1 2 2 3 2 1 3 2 2 2 1 1 2 3 2 2 1 1 3 1 1 2 3 1 2 3 1 1 1 2 1 1 3 1 1 1 2 2 3 1 3 2 1 3 1 3 1 2 1 2 3 1 2 3 1 2 1 2 2 2 3 2 2 3 2 1 2 3 2 3 2 2 2 2 1 3 1 3 2 2 2 3 1 2 2 1 3 2 1 2 3 2 2 2 3 1 1 2 1 1 3 1 3 1 2 2 3 2 3 1 2 3 1 3 1 1 1 2 1 1 1 2 3 1 1 2 1 3 1 1 2 1 3 1 3 1 1 2 3 2 1 3 1 3 2 1 3 2 1 3 2 1 1 2 2 2 3 1 1 2 3 2 2 2 3 1 1 1 3 2 3 1 3 2 1 1 2 2 3 1 2 2 3 1 2 2 3 2 2 1 1 3 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3 2 2 1 2 2 3 2 3 1 1 1 3 1 3 2 2 2 1 2 3 1 2 1 1 1 2 1 3 2 1 3 2 3 1 2 1 3 1 3 1 1 3 1 2 2 2 1 3 2 3 2 1 2 3 1 1 3 2 3 2 1 1 2 1 1 3 1 2 2 1 2 3 1 2 2 1 1 3 1 2 2 3 2 3 2 1 3 2 3 2 2 1 2 1 1 3 2 2 2 1 2 3 1 2 1 2 2 2 3 2 1 3 1 2 3 1 3 2 2 1 2 3 2 3 2 1 2 3 1 1 3 1 2 2 1 2 1 3 2 2 1 3 3 1 1 1 2 2 3 2 2 3 2 1 2 1 3 1 3 2 3 1 2 1 2 1 2 1 3 1 1 1 2 1 3 2 2 2 1 1 3 2 1 2 1 3 2 3 2 3 2 3 1 2 2 2 1 3 1 2 3 2 2 2 1 2 2 3 2 3 1 3 1 1 1 1 3 2 2 3 1 2 1 2 2 2 3 2 2 3 2 2 1 3 1 2 3 1 2 3 1 2 3 2 3 1 2 1 1 2 3 1 3 1 1 2 1 1 1 3 1 1 1 1 1 3 2 2 2 1 3 2 2 2 3 2 1 2 2 1 3 2 1 3 1 3 1 3 2 2 2 3 1 2 3 2 3 1 2 1 3 2 1 1 1 2 1 3 1 1 1 1 3 2 3 2 2 1 2 2 3 1 1 2 3 2 3 1 2 3 2 2 1 2 1 1 1 2 2 3 1 1 3 2 3 2 3 1 2 1 1 2 3 2 2 2 3 2 3 2 2 2 1 3 2 3 1 2 2 1 1 1 3 1 2 1 3 1 2 2 1 3 1 2 1 1 3 2 3 2 1 2 1 1 3 1 3 1 1 3 2 3 2 2 1 1 1 2 3 1 1 2 2 2 3 2 2 2 3 2 3 1 2 3 1 1 3 2 2 1 1 1 1 3 1 1 2 2 3 1 3 1 1 1 2 3 1 1 1 3 2 2 1 3 1 3 2 3 2 1 1 3 1 3 2 1 2 1 1 1 3 2 1 2 2 2 3 1 3 1 1 2 2 1 1 3 1 2 2 3 2 2 1 2 1 2 3 2 3 1 3 2 2 1 2 3 1 1 1 3 2 3 2 2 3 2 2 2 1 1 3 2 1 1 3 1 2 1 1 1 3 2 1 1 1 2 3 2 2 1 2 3 2 3 1 3 1 3 1 1 1 1 1 3 1 2 1 2 2 3 1 2 2 3 1 3 1 2 1 3 1 3 2 2 1 1 3 2 3 1 2 1 2 3 1 1 2 1 2 3 2 3 1 3 1 1 1 2 1 1 1 2 1 3 1 3 2 2 2 3 2 2 1 1 2 3 2 1 1 3 2 3 3 1 2 2 2 1 3 1 2 3 1 3 2 2 1 1 3 1 1 2 2 2 1 3 2 2 3 2 1 1 1 2 3 1 3 2 3 2 3 1 1 2 1 2 2 3 2 1 1 3 2 1 3 2 3 2 1 2 2 2 3 1 3 1 2 1 1 2 1 3 1 1 2 3 1 3 2 2 1 1 1 3 1 3 2 2 3 2 2 3 1 2 1 2 2 2 1 1 1 3 1 3 2 3 2 1 2 2 1 3 1 1 1 2 1 3 2 2 2 3 3 2 2 2 1 3 2 2 1 2 2 2 3 1 2 3 1 3 1 2 1 1 2 3 1 1 3 2 3 2 1 1 1 2 3 1 1 2 1 1 1 3 1 3 2 2 3 2 1 1 2 1 1 1 3 2 3 1 3 2 1 3 1 1 3 2 3 2 1 1 2 2 2 1 2 2 3 1 3 2 2 2 3 2 3 2 1 1 1 3 1 1 3 1 2 1 2 2 2 1 2 1 3 2 2 3 2 2 3 2 3 2 2 3 1 1 1 3 2 2 1 2 3 1 1 1 2 1 2 3 1 2 2 3 2 3 2 2 2 3 2 2 3 2 1 1 1 3 1 3 1 2 3 2 1 1 1 3 2 3 1 3 2 2 1 2 2 1 2 2 3 1 1 3 1 1 1 3 2 2 1 3 1 2 3 1 2 3 1 1 2 2 1 2 3 2 2 1 2 2 2 3 2 2 2 1 3 2 2 2 3 2 3 2 3 1 1 1 1 2 1 2 3 1 1 2 2 2 3 1 1 3 1 3 1 1 3 2 3 1 3 1 2 2 1 3 1 2 1 2 1 3 1 1 2 1 2 2 3 1 1 3 1 3 2 2 1 3 1 1 2 1 1 3 1 3 1 1 1 2 3 2 1 2 3 2 3 2 2 2 2 1 2 3 1 1 1 3 1 3 1 1 3 2 3 2 1 2 2 1 2 3 3 2 1 1 3 2 1 2 2 1 1 3 2 3 2 3 1 2 2 2 1 3 2 1 1 2 1 1 1 3 1 3 1 1 3 2 1 1 1 3 1 3 2 1 1 1 3 2 1 2 2 3 2 2 1 1 2 2 3 1 1 3 2 3 2 1 2 3 1 1 1 3 2 1 2 1 2 1 3 2 2 3 1 3 2 2 3 1 3 2 1 1 3 1 2 2 2 1 3 1 2 3 1 3 1 2 1 2 1 2 3 1 1 1 3 1 2 1 3 2 1 2 1 1 3 1 1 3 1 2 3 1 2 2 2 3 2 3 2 1 1 1 2 3 2 2 1 3 2 1 1 2 1 1 3 1 1 1 3 1 2 3 1 1 3 1 3 1 3 1 2 2 2 3 2 3 1 3 2 1 1 1 3 2 1 1 1 2 1 3 1 1 1 1 1 2 1 3 1 2 3 2 1 3 1 1 2 2 2 3 2 3 2 3 2 2 3 1 1 1 2 2 1 3 2 3 2 2 2 3 2 3 2 3 2 1 2 2 1 2 1 2 2 2 3 1 3 2 1 2 3 1 2 1 3 1 1 3 1 2 2 3 2 2 1 2 1 3 1 3 2 2 3 1 1 3 2 1 2 3 2 1 1 1 3 2 2 2 2 1 1 2 2 2 3 2 3 1 1 2 3 2 2 3 2 2 1 2 2 3 2 3 2 2 1 3 2 2 2 1 2 3 1 3 1 3 2 3 1 3 1 2 2 2 1 1 3 2 2 3 2 2 1 3 1 3 2 3 2 2 2 1 2 3 1 1 1 2 2 2 2 2 2 1 2 2 3 2 3 1 2 3 2 3 1 1 1 2 1 1 3 1 3 1 3 2 1 1 3 2 1 1 2 1 2 3 1 2 1 3 2 3 1 2 2 1 1 3 2 3 1 3 1 2 3 1 2 3 2 1 2 1 2 3 2 1 3 2 1 1 2 1 1 1 2 2 3 1 3 1 1 1 3 1 3 1 2 1 1 1 2 3 1 2 1 3 2 2 2 3 1 1 3 2 3 1 2 3 2 2 1 3 1 1 2 3 2 2 2 1 1 3 2 2 3 2 2 3 2 3 2 1 1 2 2 3 2 2 1 3 2 1 1 1 1 2 1 3 2 3 1 3 1 1 3 2 3 1 2 1 1 3 1 2 1 2 2 2 3 2 3 2 3 1 2 1 1 3 2 1 1 2 2 3 1 3 2 2 1 1 1 2 2 1 3 2 2 1 2 2 3 2 2 2 3 2 3 1 1 2 2 2 3 1 3 1 1 2 1 3 2 2 3 1 1 2 1 3 2 1 1 2 2 2 3 1 1 3 1 3 1 2 3 2 2 2 3 2 3 2 2 2 3 1 1 2 1 3 1 3 1 1 2 1 2 3 1 2 1 1 1 3 1 2 1 2 3 1 3 1 3 1 2 2 3 2 1 1 2 1 1 1 3 1 2 3 1 3 1 2 3 2 2 3 2 2 1 1 1 3 2 2 1 1 3 2 3 1 1 1 2 2 2 3 2 1 1 3 1 1 2 2 1 3 2 3 3 1 1 1 2 3 1 3 1 3 2 2 1 2 2 3 1 2 1 3 2 2 2 1 2 2 2 3 2 1 1 1 2 3 1 3 1 2 1 2 1 3 2 3 2 2 1 3 3 2 2 1 1 2 2 3 2 3 1 2 1 2 2 2 3 1 2 2 1 3 2 3 1 3 1 3 2 3 2 2 3 1 2 1 1 1 3 1 2 3 2 2 2 1 2 1 1 1 2 2 3 2 3 1 3 1 1 1 2 2 3 1 2 1 1 3 1 1 3 1 2 2 1 1 1 3 1 3 1 1 2 2 3 1 3 1 1 3 1 3 1 1 1 2 2 2 3 2 2 1 3 1 1 3 1 1 2 2 3 1 1 2 3 2 1 2 3 2 1 3 2 2 1 1 3 1 2 1 2 3 2 3 2 3 1 2 3 2 2 2 1 1 2 3 1 3 2 2 1 2 3 2 2 3 2 1 1 2 1 3 1 1 1 2 2 3 2 2 1 3 1 2 1 1 3 2 2 2 1 3 1 3 1 2 2 3 1 3 1 1 1 2 3 1 3 2 1 1 2 1 1 3 1 3 2 1 2 2 2 3 1 1 3 2 2 3 2 2 2 1 1 3 2 3 2 1 1 2 3 1 2 2 2 3 2 2 1 3 2 2 3 1 1 3 1 1 3 1 2 2 3 2 2 1 2 2 3 2 2 3 1 1 2 1 2 1 3 1 1 1 3 1 2 2 1 1 1 3 1 3 2 3 1 1 2 3 2 1 1 1 2 2 3 2 2 1 3 1 1 1 2 2 2 3 1 3 2 3 2 3 1 2 2 3 2 2 1 3 2 3 2 3 2 2 1 2 2 3 1 2 2 1 2 3 3 1 3 1 1 2 2 1 2 3 2 3 2 3 1 1 2 1 2 1 3 1 1 1 2 2 3 1 2 2 3 1 2 1 1 1 3 2 1 1 1 3 1 3 2 3 2 1 3 2 3 2 3 2 1 1 1 2 2 3 1 1 2 1 2 3 2 2 1 1 2 3 1 1 1 3 2 1 1 1 3 1 1 1 3 1 1 3 2 2 2 3 1 1 1 3 2 2 2 1 3 2 2 3 1 1 3 1 1 2 1 3 1 1 1 3 1 1 1 3 3 2 3 2 1 1 2 1 1 3 1 3 2 3 1 1 2 1 3 2 1 1 2 2 2 1 2 2 3 1 1 1 2 1 1 3 1 3 1 3 1 2 2 2 3 2 3 1 1 2 3 1 3 1 1 1 3 1 1 3 1 1 3 2 2 1 1 3 1 2 2 2 1 1 3 1 3 2 3 1 3 2 1 2 1 2 2 3 2 2 1 1 1 3 1 1 2 2 2 3 2 1 1 1 3 2 3 1 2 3 1 2 3 2 1 1 3 1 2 1 3 1 2 3 2 2 1 2 3 2 3 1 2 3 1 1 1 2 1 2 3 2 1 2 3 2 1 3 1 1 2 1 1 1 3 2 3 2 2 1 1 1 3 2 3 2 2 1 1 1 1 3 1 3 2 1 2 3 2 3 2 3 2 1 2 3 1 2 1 2 2 2 1 1 1 3 1 2 1 1 3 1 3 2 2 1 3 2 1 1 1 2 2 3 2 3 1 1 3 1 1 2 2 1 3 1 3 1 1 2 1 1 3 2 3 2 3 1 2 1 3 1 2 1 1 3 1 1 1 3 2 3 1 1 1 2 3 2 1 1 1 2 2 3 3 1 2 3 1 1 1 3 1 2 3 2 2 2 1 1 1 3 2 2 2 3 2 2 1 3 2 3 2 1 1 3 2 1 1 2 1 1 3 2 2 2 3 1 3 1 1 1 3 2 2 3 1 3 1 1 2 2 1 3 1 1 2 2 2 3 1 2 1 1 1 3 2 2 1 1 3 1 1 1 2 2 2 3 2 1 2 3 2 3 2 2 3 2 2 3 1 3 1 1 3 1 2 2 2 1 3 1 2 3 1 1 1 2 3 1 3 2 2 2 2 1 1 3 2 2 2 3 1 3 1 2 1 1 1 3 1 2 3 1 2 1 2 3 2 3 1 3 1 2 1 3 2 2 2 3 2 1 1 2 1 2 3 2 2 2 3 2 1 3 2 2 2 3 1 1 1 2 2 3 2 1 1 3 2 2 2 3 1 2 3 1 2 1 3 1 1 2 2 3 1 2 2 1 1 2 3 1 2 3 1 3 2 1 3 2 1 3 1 3 1 2 2 2 3 2 1 1 2 1 1 3 2 1 2 2 3 1 1 3 1 2 1 1 2 3 1 2 3 2 1 1 2 3 2 1 1 3 2 1 3 2 3 2 2 3 1 1 1 2 2 2 3 1 2 3 1 3 1 3 1 2 1 2 3 2 2 1 2 3 2 3 2 1 1 1 3 2 1 2 1 3 2 2 2 1 2 3 2 2 1 3 2 3 1 1 2 1 1 3 2 3 1 1 1 2 1 3 1 1 2 3 1 1 2 3 1 1 1 3 1 1 1 3 1 2 2 3 2 1 1 2 1 1 3 2 1 3 1 3 1 1 2 3 1 1 1 2 1 3 2 3 2 2 1 1 1 2 3 1 3 2 3 2 3 2 1 3 1 2 1 1 1 3 1 2 3 2 3 1 1 2 2 1 2 3 1 2 3 1 2 1 3 2 1 2 1 2 3 2 3 2 3 2 1 2 2 2 3 2 2 2 1 2 3 2 2 2 3 2 1 3 1 1 1 2 3 2 2 2 3 1 1 3 1 2 1 1 1 2 2 2 3 2 1 3 1 3 1 3 1 1 1 2 2 2 3 2 2 3 2 1 3 1 1 2 1 1 3 1 2 2 1 3 2 1 1 3 2 3 2 1 3 1 2 3 1 2 2 2 1 3 1 3 1 1 1 2 1 2 3 1 3 2 1 3 1 1 1 1 2 1 3 1 3 2 1 2 3 2 2 3 2 2 2 1 2 3 1 3 1 1 3 1 2 3 1 2 3 1 1 3 1 3 2 2 2 1 2 2 3 2 1 1 1 2 1 1 3 2 1 1 1 3 1 1 3 1 1 3 1 1 1 2 3 2 3 2 2 1 2 2 3 1 1 3 1 1 2 2 1 1 3 2 3 2 2 2 1 3 2 3 2 1 1 3 1 1 1 3 1 1 2 3 2 2 3 1 2 2 2 1 2 3 1 2 3 2 3 1 2 2 1 1 1 3 1 3 1 2 3 2 2 3 1 2 2 3 1 1 1 2 1 1 2 3 1 2 1 1 2 2 3 2 2 3 1 3 1 3 1 3 2 1 1 2 3 2 2 2 3 2 2 3 1 1 1 3 2 3 2 1 1 1 3 2 1 2 1 2 2 3 1 3 2 2 1 2 1 2 3 1 3 1 1 1 3 2 3 2 1 1 2 2 2 2 3 2 3 1 3 1 1 1 3 1 1 3 2 1 2 1 2 1 3 1 1 2 3 2 1 1 3 2 2 2 1 3 1 3 2 2 1 2 1 3 1 3 2 2 2 1 3 1 2 1 3 1 2 1 3 1 2 1 1 3 2 2 1 1 2 2 3 1 1 3 1 3 1 3 1 2 3 1 2 2 3 2 2 2 1 2 3 2 1 2 2 1 2 3 1 1 1 3 2 2 1 1 3 1 1 1 2 2 3 2 1 3 2 3 1 2 1 3 2 2 2 3 1 2 1 2 2 3 2 2 2 3 2 3 1 3 2 3 2 1 2 1 1 2 2 2 3 2 1 3 1 1 1 3 2 2 3 2 2 1 2 3 1 3 2 2 3 1 2 2 2 3 1 3 2 1 1 3 2 2 2 1 2 1 3 1 2 3 1 1 1 1 3 1 2 1 1 1 3 2 3 1 3 2 2 3 1 2 2 2 1 3 1 2 3 1 2 1 2 2 3 2 1 1 3 1 2 1 2 3 2 2 3 2 1 1 1 3 3 2 1 1 3 1 3 2 3 2 1 2 2 3 2 1 1 3 2 2 1 1 2 2 3 2 3 2 3 1 2 2 1 3 2 1 1 2 3 1 1 3 2 1 2 2 2 1 3 2 1 1 3 1 1 1 3 1 2 2 1 1 3 2 3 2 2 1 3 2 1 1 1 3 2 1 3 1 1 1 3 2 2 3 1 1 1 2 2 3 1 2 2 1 2 3 2 1 1 3 1 3 1 1 3 2 2 3 1 3 2 1 1 2 3 2 1 2 2 2 3 2 2 1 1 3 1 1 1 2 1 3 2 1 3 1 2 1 1 3 2 3 1 1 2 1 1 3 2 1 1 1 2 2 3 1 1 1 3 2 3 2 1 2 1 3 2 3 1 1 3 1 2 3 2 1 2 3 2 2 2 1 2 2 3 2 2 3 2 3 2 1 1 2 2 2 1 3 1 1 2 1 2 1 3 2 3 1 1 3 1 3 1 2 1 3 3 2 2 1 2 3 1 1 1 3 1 3 2 1 2 3 2 3 2 2 1 1 1 2 2 1 2 2 1 2 3 2 3 1 1 3 1 1 3 1 1 2 3 1 2 2 1 3 2 1 1 2 1 1 3 2 2 3 1 1 3 1 3 1 1 2 2 3 2 2 3 2 2 3 1 2 3 2 2 2 3 1 2 3 2 1 1 2 2 3 2 2 1 1 1 3 3 2 3 1 1 1 3 1 2 2 2 3 1 3 2 2 2 3 2 1 2 1 1 2 1 3 1 3 1 1 2 1 2 1 3 1 2 2 3 1 3 1 2 2 2 3 2 2 2 2 2 3 1 3 1 2 3 2 3 1 2 3 1 2 1 1 1 3 2 2 1 1 3 2 2 3 2 1 1 1 2 2 3 2 1 3 2 1 1 1 3 1 1 3 2 1 3 2 3 2 2 1 2 3 1 2 3 2 2 3 2 2 2 3 2 1 2 2 1 2 1 2 2 1 2 2 3 2 3 2 1 3 1 2 3 2 1 2 2 1 1 3 1 3 3 2 2 1 3 1 1 1 3 1 2 2 2 1 3 1 1 3 2 2 1 3 2 2 2 2 3 2 3 2 1 2 2 1 1 3 1 3 1 3 2 3 1 1 1 2 1 2 3 2 2 2 1 1 3 1 2 1 3 1 1 1 3 1 3 2 3 1 2 2 2 1 1 1 2 3 1 3 1 1 1 2 1 3 1 2 1 3 2 2 1 2 2 3 2 3 2 3 1 1 2 2 3 1 1 2 1 1 3 1 1 2 2 2 3 2 2 3 2 3 1 1 2 1 1 3 1 2 2 3 1 1 2 2 1 3 2 3 1 3 2 1 1 3 1 1 2 3 2 2 2 3 1 3 1 3 1 2 2 2 1 3 2 1 1 1 3 1 1 3 2 2 2 1 3 1 1 2 1 3 1 1 1 2 3 2 3 2 2 2 3 1 2 1 2 1 1 3 2 1 1 3 2 3 2 2 1 1 3 1 2 2 2 3 1 3 3 2 1 3 2 3 1 1 2 1 1 3 2 2 1 3 2 3 2 2 1 1 2 1 1 1 3 2 3 2 3 2 2 1 1 1 3 2 1 1 1 2 3 2 1 3 1 2 1 3 1 3 1 2 3 2 2 2 1 2 3 2 2 3 2 3 1 1 2 2 1 1 1 3 2 2 3 1 1 2 1 2 2 3 1 2 3 1 2 1 1 3 1 1 3 1 2 3 1 1 2 3 2 3 1 3 1 2 3 2 2 2 1 3 1 1 2 1 1 2 1 1 2 1 1 2 3 1 2 3 2 1 1 3 2 2 2 3 1 3 2 2 2 3 1 1 1 3 1 3 2 3 1 1 2 1 3 1 1 1 2 1 1 3 1 3 1 1 1 1 2 1 1 1 3 2 2 1 2 2 3 1 3 1 3 1 3 2 2 2 1 3 1 3 2 1 3 2 3 2 2 3 2 1 3 2 2 2 1 3 2 1 2 1 2 1 3 2 1 1 3 1 1 2 3 2 1 2 2 1 3 1 2 1 2 2 2 3 2 3 3 1 2 1 1 1 2 3 2 2 2 3 1 2 1 1 1 3 2 1 3 2 2 3 1 2 1 3 2 1 2 3 2 1 2 3 2 3 2 3 1 1 3 1 2 2 2 1 1 2 3 1 1 2 3 2 1 3 1 3 2 3 1 2 2 1 3 2 2 2 1 1 3 2 1 3 2 1 2 2 2 1 3 2 3 1 2 3 2 1 1 3 1 1 2 1 1 3 1 1 2 2 3 2 1 2 2 3 1 1 3 1 1 3 1 1 2 1 2 3 2 2 2 1 2 1 3 1 1 2 2 3 1 3 1 3 1 1 3 2 2 1 1 3 1 1 1 3 2 1 3 2 1 3 1 3 1 2 2 2 3 1 3 1 1 2 2 1 2 2 2 1 1 1 3 1 1 1 3 2 1 2 2 3 2 1 1 3 1 3 2 3 1 1 1 2 2 3 1 3 1 1 1 3 2 3 1 1 2 3 1 1 3 2 2 2 1 1 3 1 1 1 2 1 1 3 2 1 2 3 1 2 1 3 2 1 3 2 1 3 1 2 2 2 3 1 1 2 2 3 2 1 2 2 3 2 1 3 2 2 2 3 2 3 1 1 3 1 3 1 1 2 1 1 2 3 2 1 3 1 3 1 2 1 2 1 1 3 2 3 2 3 2 1 1 2 1 3 2 2 3 2 2 1 1 2 3 1 3 2 1 1 2 1 2 1 3 2 2 3 2 1 3 2 2 2 1 3 1 2 3 1 1 2 3 2 1 2 2 3 2 3 2 2 1 3 1 1 2 3 1 2 3 2 2 1 1 2 1 3 3 2 2 2 3 2 1 2 1 3 2 1 2 2 2 3 1 2 2 3 1 2 3 2 1 3 1 3 2 1 1 1 3 2 1 2 3 1 3 2 2 1 2 3 1 1 2 1 3 1 1 1 3 2 2 2 1 1 3 2 3 1 2 3 2 1 2 1 2 2 3 2 2 2 1 1 1 2 3 1 2 1 1 1 3 1 3 2 1 3 2 3 1 1 3 2 2 2 1 1 1 2 3 2 3 2 3 1 3 1 1 3 1 2 3 1 1 2 1 1 1 2 2 2 3 2 1 2 1 1 1 3 2 3 1 1 3 1 1 3 1 3 1 1 2 3 1 2 2 1 3 2 1 2 2 2 3 2 3 1 1 3 1 3 1 2 2 2 2 2 2 3 1 1 2 3 1 1 1 2 2 3 1 2 3 1 2 1 3 1 2 3 1 3 1 3 2 1 1 3 1 2 2 1 1 3 1 1 2 1 1 3 1 1 1 3 1 2 2 3 1 1 2 2 3 1 3 1 1 3 2 3 1 1 3 2 1 1 1 2 2 2 2 1 3 1 3 1 1 3 2 1 2 2 3 2 2 2 3 1 1 1 3 1 2 1 1 1 3 2 3 1 1 1 3 1 2 2 2 3 1 1 1 2 3 1 2 3 3 1 1 1 3 2 2 1 3 1 3 1 1 1 2 3 2 1 3 1 1 1 2 2 3 2 3 1 1 2 1 1 2 3 1 1 3 1 1 3 2 2 1 2 3 2 2 1 2 2 3 2 3 1 1 2 1 1 1 3 2 1 3 1 2 3 2 3 2 2 1 2 2 2 1 2 1 2 3 1 2 1 2 3 1 3 2 2 2 3 2 3 2 2 3 1 2 2 3 1 2 2 2 3 2 3 2 3 1 3 2 1 2 2 1 3 2 2 1 2 1 1 1 3 2 3 1 2 2 1 1 3 2 2 1 3 2 2 2 3 1 3 1 2 2 2 3 2 1 2 2 2 3 2 1 2 1 1 2 3 2 2 3 1 1 3 1 3 3 2 2 2 3 1 1 1 2 2 1 3 2 3 2 3 1 3 1 1 1 2 1 2 1 1 2 3 2 2 3 1 3 1 2 2 3 1 2 1 1 2 3 2 2 3 1 1 2 1 3 2 1 3 2 1 3 2 1 2 2 3 2 2 3 2 1 1 2 1 1 3 3 2 2 3 2 1 1 2 2 2 3 1 3 2 3 2 2 1 3 2 2 1 2 2 2 3 1 1 2 1 2 3 1 2 1 3 2 2 1 3 2 1 1 2 2 3 2 3 2 3 1 2 1 3 2 1 2 3 2 2 2 3 2 3 1 2 2 1 1 1 3 1 3 1 2 3 2 1 2 1 1 1 3 1 3 2 1 2 3 2 2 1 2 1 1 3 1 3 2 3 1 3 1 2 2 2 1 3 1 1 3 1 2 3 2 2 1 2 2 1 1 2 3 1 3 1 1 2 2 2 3 2 2 1 1 1 3 1 3 1 1 1 3 2 1 1 1 3 1 1 2 2 1 3 2 1 2 3 1 2 1 3 1 2 3 1 3 1 2 1 3 1 3 2 2 3 2 1 2 1 3 2 2 2 1 2 1 3 2 2 3 1 3 2 1 3 1 1 2 3 1 2 2 3 2 2 2 1 3 1 1 3 1 2 2 2 3 2 2 2 1 2 3 2 2 2 3 1 3 1 1 3 1 3 2 2 1 2 2 2 2 1 3 1 1 3 2 2 2 3 1 1 1 3 2 2 1 2 2 3 1 2 2 3 3 1 2 3 1 1 3 1 3 2 1 2 2 2 3 2 2 1 2 1 2 3 2 1 3 1 2 3 1 1 2 1 2 1 3 2 1 1 3 2 1 2 2 3 1 3 2 1 2 1 3 2 3 1 2 3 1 1 1 2 2 2 3 1 3 1 2 1 3 1 2 1 3 1 1 1 3 1 1 1 2 2 3 1 1 3 1 3 2 2 2 3 1 2 1 2 1 2 2 2 3 1 3 2 1 2 2 2 3 2 3 2 1 2 2 3 1 1 2 3 1 2 3 1 3 2 2 3 1 1 1 2 2 2 3 1 1 3 2 1 2 2 3 2 2 2 1 1 2 1 3 2 3 1 3 1 3 1 3 2 1 2 1 2 3 2 1 1 1 2 2 1 1 3 1 3 1 3 2 3 1 3 2 1 1 1 2 3 2 1 1 1 1 1 3 1 1 2 1 3 1 2 3 1 3 1 2 2 1 3 1 1 1 2 1 3 1 3 2 2 2 1 1 1 3 1 3 2 2 1 3 1 1 2 2 3 1 1 1 3 3 2 1 1 3 1 2 2 2 3 2 2 3 1 1 2 1 1 1 3 1 1 3 1 1 3 1 3 1 1 1 3 1 1 3 2 2 1 1 1 3 2 3 1 2 1 2 2 2 1 1 2 1 3 1 3 1 1 3 1 3 1 2 3 2 1 2 3 1 1 2 1 2 2 1 2 2 1 3 2 3 1 2 1 1 3 2 3 1 1 3 2 2 2 1 3 1 2 1 1 2 3 2 1 1 1 3 1 2 3 1 3 2 2 2 1 2 3 1 3 2 2 3 1 2 2 2 3 1 3 1 3 2 2 3 1 2 1 1 3 1 2 2 2 1 2 3 1 2 2 1 2 2 3 2 3 2 3 2 1 3 1 1 2 2 1 3 1 2 1 2 1 1 1 3 1 2 1 2 1 3 2 1 3 1 2 3 1 2 3 2 3 2 2 2 1 3 2 2 3 1 3 1 2 3 1 1 3 2 2 1 2 2 1 3 1 1 2 2 3 1 1 2 2 3 1 2 1 2 1 3 2 3 2 1 1 1 3 2 3 3 1 1 3 1 1 1 3 1 2 2 1 2 2 3 2 1 2 2 3 1 3 2 2 1 2 2 3 1 3 2 3 2 1 3 2 3 1 2 2 2 1 3 1 1 1 2 1 1 1 2 1 1 1 3 2 3 2 2 2 1 1 3 1 3 2 1 3 1 3 2 1 3 2 1 3 1 3 1 2 1 1 2 2 3 1 2 3 2 3 2 1 1 2 2 2

wherein each of 1 to 3 is a nucleotide base selected to be different from the others of 1 to 3 with the proviso that up to three nucleotide bases of each sequence can be substituted with any nucleotide base provided that: for any pair of sequences of the set: M1≦16, M2≦13, M3 S 20., M4 s 16, and M5≦19, where: M1 is the maximum number of matches for any alignment in which there are no internal indels; M2 is the maximum length of a block of matches for any alignment; M3 is the maximum number of matches for any alignment having a maximum score; M4 is the maximum sum of the lengths of the longest two blocks of matches for any alignment of maximum score; and M5 is the maximum sum of the lengths of all the blocks of matches having a length of at least 3, for any alignment of maximum score; wherein the score of an alignment is determined according to the equation (A×m)−(B×mm)−(C×(og+eg))−(D×eg)), wherein: for each of (i) to (iv):  (i) m=6, mm=6, og=0 and eg=6,  (ii) m=6, mm=6, og=5 and eg=1,  (iii) m=6, mm=2, og=5 and eg=1, and  (iv) m=6, mm=6, og=6 and eg=0, A is the total number of matched pairs of bases in the alignment; B is the total number of internal mismatched pairs in the alignment; C is the total number of internal gaps in the alignment; and D is the total number of internal indels in the alignment minus the total number of internal gaps in the alignment; and wherein the maximum score is determined separately for each of (i), (ii), (iii) and (iv) b) mixing said cleavage means, said target nucleic acid, said first and second oligonucleotides to create a reaction mixture under reaction conditions such that at least said 5′ portion of said first oligonucleotide is annealed to said target nucleic acid and wherein at least said 5′ and central portion of said second oligonucleotide is annealed to said target nucleic acid so as to create a cleavage structure and wherein the combined melting temperature of said complementary regions within said 5′ and 3′ portions of said first oligonucleotide when annealed to said target nucleic acid is greater than the melting temperature of said 5′ and central portion of said first oligonucleotide, and wherein cleavage of said cleavage structure occurs to generate non-target cleavage products; and c) detecting said non-target cleavage products.
 36. The composition of claim 35, wherein one or more of said first and second oligonucleotides contain a 3′-terminal dideoxynucleotide.
 37. The composition of claim 35, wherein the composition includes a plurality of said target nucleic acid molecules and a plurality of said second oligonucleotide molecules such that each of said second oligonucleotide molecules has a distinct 3′ region.
 38. The composition of claim 35, wherein the composition includes at least ten, or twenty, or thirty, or forty, or fifty, or sixty, or seventy, or eighty, or ninety, or one hundred, or one hundred and ten, or one hundred and twenty, or one hundred and thirty, or one hundred and forty, or one hundred and fity, or one hundred and sixty said second oligonucelotide molecules, or comprising one hundred and seventy said second oligonucleotide molecules, or comprising one hundred and eighty said second oligonucleotide molecules, or comprising one hundred and ninety said second oligonucleotide molecules, or comprising two hundred said second oligonucleotide molecules, or comprising two hundred and twenty said second oligonucleotide molecules, or comprising two hundred and forty said second oligonucleotide molecules, or comprising two hundred and sixty said second oligonucleotide molecules, or comprising two hundred and eighty said second oligonucleotide molecules, or comprising three hundred said second oligonucleotide molecules, or comprising four hundred said second oligonucleotide molecules, or comprising five hundred said second oligonucleotide molecules, or comprising six hundred said second oligonucleotide molecules, or comprising seven hundred said second oligonucleotide molecules, or comprising eight hundred said second oligonucleotide molecules, or comprising nine hundred said second oligonucleotide molecules, or comprising one thousand said second oligonucleotide molecules, or comprising eleven hundred said second oligonucleotide molecules.
 39. A method of detecting the presence of a target nucleic acid molecule by detecting non-target cleavage products, the method comprising: a) providing: i) a cleavage means, ii) a target nucleic acid, said target nucleic acid having a first region, a second region and a third region, wherein said first region is located adjacent to and downstream from said second region, and said second region is located adjacent to and downstream from said third region; iii) a first oligonucleotide having a 5′ and a 3′ portion, said 5′ portion of said first oligonucleotide having a sequence complementary to said second region of said target nucleic acid and said 3′ portion of said first oligonucleotide having a sequence complementary to said third region of said target nucleic acid; iv) a second oligonucleotide having a 5′ portion, a central portion and a 3′ portion, said 5′ portion of said second oligonucleotide having a sequence complementary to said first region of said target nucleic acid, said central portion of said second oligonucleotide having a sequence complimentary to said second region of said target nucleic acid, and said 3′ portion of said second oligonucleotide having a sequence that is not base-paired to said target nucleic acid and is selected from a set of oligonucleotides based on a following group of sequences, 1 1 1 2 2 3 2 3 1 1 1 3 1 2 2 3 2 2 2 3 2 3 2 1 3 2 2 1 3 1 3 2 2 1 1 2 2 3 2 1 2 2 2 3 1 2 3 1 1 2 3 2 2 1 1 1 3 2 1 1 3 2 3 2 2 3 1 1 1 2 3 2 2 3 1 2 3 2 2 1 3 1 1 3 2 1 2 1 2 2 3 2 3 1 1 2 2 2 2 3 2 3 2 1 3 1 1 2 1 2 3 2 3 2 2 3 2 2 1 1 1 2 1 1 3 2 3 2 1 1 3 2 3 1 1 1 2 1 1 3 1 1 3 1 1 1 3 1 3 2 1 2 2 2 3 2 2 3 2 3 1 3 2 2 1 1 1 2 3 2 3 2 2 2 1 2 3 2 2 1 2 1 2 3 2 3 1 1 3 2 2 2 1 1 1 3 1 3 1 1 2 1 3 1 1 2 1 2 3 2 3 2 1 1 3 2 2 1 2 3 1 1 1 3 1 3 2 3 1 3 1 2 1 1 2 3 2 2 2 1 1 2 3 1 3 1 1 1 2 1 2 3 2 2 1 3 1 1 2 3 2 3 1 2 2 2 1 3 2 2 3 2 2 3 1 2 3 2 2 2 1 3 2 1 3 2 2 2 3 2 1 1 1 3 1 3 2 1 2 1 1 3 2 2 2 3 1 2 3 1 2 1 1 1 1 3 2 1 1 3 1 1 2 3 1 2 3 2 1 1 2 1 1 3 2 3 3 2 1 3 1 1 1 2 1 3 2 2 2 1 2 2 3 1 2 3 1 2 2 3 2 3 2 1 1 3 2 3 1 1 1 2 1 3 2 3 1 3 2 2 1 2 2 2 1 1 1 2 1 3 1 2 3 1 2 1 2 1 1 3 2 3 1 3 1 1 2 3 1 2 1 1 3 2 2 1 2 1 1 3 2 3 2 2 1 2 3 2 3 1 3 2 2 1 2 1 3 1 2 1 1 1 3 1 3 1 2 3 1 2 2 2 3 2 2 3 1 3 1 3 2 2 3 1 3 1 1 2 3 2 1 2 1 3 2 1 2 2 1 2 1 1 3 2 1 3 2 2 2 3 2 1 1 3 1 1 2 3 1 2 2 3 2 1 2 2 1 2 3 1 1 1 2 2 3 1 3 2 3 1 1 3 1 2 2 3 1 2 3 2 1 2 1 2 3 2 1 1 1 2 2 3 2 2 1 2 3 2 2 3 1 3 3 1 1 2 2 3 2 1 2 1 1 1 3 2 1 2 2 1 3 1 2 3 2 3 2 1 3 1 2 3 1 3 1 2 2 1 1 3 2 3 2 2 1 2 2 2 3 1 3 2 2 1 1 3 2 2 2 3 2 2 2 1 2 3 2 1 2 1 3 1 1 3 3 1 3 2 1 2 2 1 3 2 1 1 1 3 2 3 1 2 1 2 3 1 2 1 3 2 3 1 1 2 3 1 2 2 2 1 3 2 1 1 1 2 3 1 2 2 3 1 3 1 2 2 3 1 1 3 2 2 1 2 1 3 1 1 1 2 3 1 2 2 1 3 1 3 2 3 1 2 1 1 1 2 3 2 2 1 3 2 2 3 1 1 2 2 3 2 2 1 2 1 2 1 3 2 1 1 1 2 3 2 2 2 3 2 3 2 3 2 2 3 2 2 1 1 3 2 3 2 2 1 3 2 2 1 2 2 2 3 2 2 3 2 1 3 3 2 1 3 2 1 1 2 1 2 3 1 1 3 2 3 1 3 1 1 2 1 2 1 2 1 3 2 3 2 1 2 1 3 1 1 2 3 2 1 3 1 2 2 2 1 3 2 2 2 3 2 1 3 1 2 2 1 3 1 2 3 2 3 2 2 2 3 2 1 1 1 2 1 3 2 1 2 1 3 1 3 2 1 3 1 3 1 2 3 1 2 1 2 2 2 1 2 2 3 2 3 1 1 1 3 1 1 1 3 1 3 1 1 3 1 1 1 2 2 2 3 2 3 1 3 1 1 2 2 1 1 3 1 2 2 1 1 3 1 1 2 3 2 1 2 1 2 2 1 3 2 2 1 1 3 1 1 1 3 1 1 3 1 3 2 2 3 2 2 3 2 1 3 2 2 3 1 3 1 1 1 2 1 2 3 2 1 3 2 2 2 2 1 3 1 3 2 2 3 2 2 1 1 1 3 1 3 2 3 2 1 1 1 2 1 3 2 2 1 2 3 1 2 3 2 3 2 1 2 1 1 3 2 1 1 2 1 2 3 2 2 2 3 2 2 1 3 1 1 2 3 1 3 1 1 3 1 2 2 2 1 2 3 1 3 2 1 2 1 3 2 2 2 1 1 1 3 1 1 3 2 1 3 2 1 3 1 3 2 3 1 3 1 2 1 2 1 3 1 2 2 2 1 3 1 1 1 3 2 1 1 2 2 3 2 2 2 1 2 1 3 2 3 1 1 3 2 3 1 1 2 1 3 2 1 1 1 3 2 1 1 3 2 1 3 2 1 1 2 1 3 2 3 2 3 2 2 1 1 1 2 2 2 3 2 3 1 3 2 2 1 2 3 1 1 1 3 1 2 1 1 3 1 3 1 1 1 3 2 1 3 1 3 1 1 2 1 1 1 3 1 2 1 1 3 1 1 1 2 2 2 1 1 3 1 2 2 3 2 2 1 1 3 1 3 2 1 3 1 1 3 3 2 2 2 1 1 1 3 1 2 2 3 2 1 1 3 1 1 2 3 2 3 2 1 2 2 2 3 2 3 1 1 3 1 2 3 1 1 3 2 1 2 2 2 3 2 1 2 2 3 2 3 2 2 2 1 3 1 1 2 2 2 1 3 2 1 2 3 2 3 2 1 3 1 2 1 1 2 3 1 2 2 1 2 1 3 1 1 1 3 2 3 2 2 2 3 3 2 2 1 2 2 2 3 2 1 1 3 2 2 1 1 3 1 2 1 3 2 1 3 1 3 2 2 2 1 2 2 3 1 1 1 3 1 3 2 2 2 3 1 1 2 1 3 2 2 3 2 3 2 2 2 1 2 2 3 2 3 2 1 3 2 2 2 1 1 1 3 1 2 2 3 2 3 1 3 1 1 3 1 2 1 2 3 1 1 1 3 2 2 1 2 2 3 1 3 1 1 2 3 2 1 1 1 3 1 1 2 3 2 2 2 1 2 2 3 1 2 3 2 3 1 1 1 3 2 2 1 2 3 1 2 3 2 2 1 1 2 2 3 3 2 2 2 1 3 2 1 2 2 1 3 2 2 3 2 2 1 1 3 1 2 2 3 3 1 2 2 3 1 2 1 2 2 2 3 1 1 2 3 2 2 2 3 2 2 2 3 2 3 1 1 2 2 3 1 1 1 3 2 3 2 1 1 2 3 2 2 3 2 1 2 3 1 2 2 3 2 1 2 2 3 2 2 3 1 3 1 1 2 1 3 1 1 2 1 1 1 1 2 2 2 3 1 3 1 2 2 2 3 2 3 1 2 1 3 1 3 2 1 3 2 1 1 2 2 1 3 1 2 2 2 3 2 2 2 3 2 2 3 2 2 3 2 3 2 2 2 3 2 1 2 2 3 2 2 1 3 2 3 1 1 2 1 2 1 3 2 1 2 3 2 1 3 2 1 3 2 1 3 1 2 3 2 2 2 1 2 3 1 1 2 2 3 2 2 2 1 1 1 3 1 2 3 1 2 2 3 1 1 3 1 1 1 2 3 2 3 2 3 1 2 1 1 2 3 1 2 3 2 2 1 2 2 2 3 2 3 2 1 1 2 1 3 2 2 3 2 3 1 3 1 1 2 2 2 3 2 1 1 2 2 1 3 1 2 1 3 1 2 3 2 1 1 3 1 3 1 1 1 2 2 3 2 3 1 1 1 1 3 1 2 2 1 1 3 1 3 1 1 3 2 2 1 1 2 1 3 1 3 2 1 3 1 1 3 2 1 1 1 2 2 3 2 3 1 1 2 3 1 1 1 3 1 1 1 1 1 2 3 2 1 1 3 1 1 1 3 1 1 3 1 2 2 3 2 2 3 2 1 2 2 2 3 1 2 2 2 1 2 3 2 3 2 2 1 2 3 2 2 3 1 3 2 3 2 1 2 2 3 1 3 1 1 1 2 2 2 3 1 1 3 1 1 2 3 1 1 3 1 1 2 2 3 2 1 2 3 1 1 1 2 3 1 1 2 2 3 2 1 1 3 2 1 2 2 3 2 1 3 1 1 3 2 1 1 1 3 2 2 1 3 1 1 3 2 2 2 2 1 2 3 2 1 1 2 3 1 2 1 1 3 2 3 2 1 3 2 2 3 1 2 1 2 1 3 2 2 3 1 1 1 2 2 3 2 3 1 2 1 3 2 3 2 1 2 1 1 3 1 1 1 2 2 1 3 1 3 1 3 2 2 3 2 1 1 1 3 3 1 1 2 2 3 2 3 1 1 1 2 3 2 3 1 2 2 3 1 2 1 2 1 1 1 1 2 1 1 3 2 1 3 2 2 2 1 1 2 3 1 3 1 3 1 1 3 3 1 2 2 1 1 1 3 1 1 3 2 1 1 3 2 3 1 1 2 3 2 2 2 2 1 2 3 2 3 2 3 2 2 3 2 2 2 1 3 2 3 2 2 1 2 2 1 3 1 3 2 2 1 2 1 2 3 2 1 3 2 2 1 3 1 3 2 2 1 2 1 3 1 1 1 3 1 1 1 3 1 1 3 2 3 2 2 1 1 3 2 2 1 1 1 2 1 3 2 1 2 2 1 3 2 1 1 3 2 1 2 3 2 3 1 2 2 3 2 2 2 3 2 3 2 3 1 2 2 3 1 1 2 1 2 2 3 2 3 1 1 1 2 1 2 3 2 3 1 1 1 3 1 3 2 2 1 1 3 2 3 1 2 2 1 1 1 3 1 2 2 3 1 1 2 3 1 2 2 3 1 3 1 2 1 2 3 2 1 1 1 1 1 3 1 2 3 1 2 1 3 2 2 1 1 3 2 3 2 1 1 3 2 2 1 2 1 3 2 2 3 2 2 1 2 2 3 1 3 1 1 2 2 2 1 3 1 1 3 2 2 2 1 2 1 3 2 3 1 1 2 2 1 2 3 1 3 2 3 1 1 1 3 3 1 2 1 3 1 2 2 2 1 3 1 1 2 3 1 1 2 2 1 1 3 2 3 2 2 2 3 1 1 3 1 1 3 1 3 1 2 2 2 3 1 1 1 2 2 3 1 1 2 3 1 1 2 1 1 3 1 3 2 2 3 1 2 1 1 1 2 3 2 3 1 2 3 2 2 2 1 2 3 2 1 3 2 3 2 1 3 1 2 2 3 1 1 2 2 2 2 2 1 1 3 2 3 1 3 2 2 1 2 1 3 1 1 3 2 1 3 2 1 3 1 2 2 2 1 2 3 2 3 2 2 2 3 1 1 3 2 2 1 1 3 1 2 2 1 3 2 2 1 3 1 3 1 1 1 3 2 3 1 2 1 1 1 3 2 2 1 3 2 1 1 2 3 1 2 1 1 2 3 1 1 3 2 3 2 1 2 1 2 1 3 1 1 2 3 1 1 3 2 3 2 2 1 3 2 1 2 1 3 1 2 1 3 2 1 2 1 1 1 2 2 3 1 3 2 2 2 3 2 2 2 3 1 2 2 3 2 1 3 2 1 1 2 3 1 1 3 1 1 2 1 1 3 2 1 2 3 1 3 2 3 2 2 1 1 1 2 3 2 1 1 2 1 3 2 3 2 2 3 2 2 1 3 2 2 1 3 1 3 1 3 2 2 1 3 2 3 1 1 1 2 3 2 2 3 2 2 1 1 1 2 3 1 1 1 2 1 3 1 1 1 2 3 2 1 2 2 3 2 2 2 3 2 3 1 1 3 2 2 1 2 1 1 3 2 2 2 3 2 3 1 3 1 1 2 2 1 1 3 3 1 3 2 2 2 1 2 1 3 2 2 1 3 1 1 2 1 2 3 2 2 3 2 1 3 1 3 2 2 1 2 2 1 3 1 1 3 1 1 3 1 2 2 2 1 1 3 3 1 3 2 2 1 1 2 3 1 1 1 2 1 1 3 2 1 2 2 2 3 2 3 1 2 3 1 2 3 1 1 2 1 3 2 2 3 1 1 3 2 1 2 1 2 1 3 1 2 1 3 1 2 1 2 3 1 3 1 2 3 1 1 1 3 2 2 1 3 2 1 2 1 2 3 2 1 1 1 3 1 1 1 3 2 3 1 1 1 3 1 1 3 1 1 2 3 1 1 2 3 2 1 3 1 1 1 2 3 1 1 2 3 2 2 3 1 1 1 1 1 2 2 3 1 1 2 1 3 2 3 2 3 2 3 1 3 2 2 2 1 1 2 1 3 1 2 1 2 2 3 2 2 2 3 1 2 2 1 1 2 3 1 1 3 1 3 1 1 1 3 2 2 3 2 1 1 1 3 2 2 3 1 1 3 1 2 1 1 1 3 3 2 2 1 1 3 1 3 1 2 2 1 2 3 1 3 1 2 3 2 1 2 2 1 1 3 1 1 3 1 2 1 2 1 1 3 1 1 3 1 2 2 3 1 1 2 2 3 3 2 1 3 1 1 1 2 2 2 3 1 1 2 2 3 1 2 3 2 3 1 1 1 1 1 3 1 3 2 1 3 1 2 2 3 1 2 1 1 3 2 1 2 1 2 3 1 2 3 1 2 1 2 1 3 2 1 3 2 3 1 1 3 1 1 1 2 1 1 3 2 1 3 1 2 1 1 2 3 1 2 3 1 3 1 1 1 2 3 1 1 3 1 2 1 1 2 3 2 3 1 1 1 3 2 1 2 2 2 3 2 3 1 2 1 2 1 3 2 1 1 2 1 1 3 1 3 1 1 2 2 3 1 2 1 2 3 1 1 3 1 2 3 2 1 1 3 2 3 2 1 2 2 2 1 3 2 1 3 1 1 2 3 1 1 3 2 2 1 2 3 2 2 1 3 1 2 2 2 3 2 2 3 1 3 1 2 2 3 1 2 1 3 2 2 2 3 2 1 2 3 1 1 3 1 3 1 2 1 3 2 1 2 2 2 3 1 3 1 1 1 2 3 2 2 1 2 3 2 1 2 2 2 1 3 2 1 3 2 2 1 2 3 2 3 1 3 1 1 2 3 2 3 2 2 2 3 1 2 2 2 1 1 3 2 1 2 3 2 2 2 3 2 2 2 1 2 1 3 1 1 2 3 2 1 2 3 3 1 3 2 1 2 1 2 1 3 1 1 3 1 1 1 3 1 1 1 2 2 2 3 1 2 3 1 3 2 3 1 1 3 2 1 1 1 2 3 2 1 3 2 2 1 2 2 2 2 1 1 3 1 1 3 2 3 1 3 2 2 1 2 2 3 2 3 1 2 1 2 1 2 3 1 1 1 2 3 1 3 1 1 2 1 2 2 3 2 2 3 2 2 2 3 3 1 2 2 1 1 2 3 1 2 2 1 2 3 2 3 1 1 2 2 3 1 2 3 3 1 1 1 2 3 2 2 1 1 1 3 1 2 1 2 3 1 1 1 3 2 1 3 2 1 2 2 3 2 2 3 1 2 2 2 3 1 2 1 2 2 1 3 2 3 2 3 2 2 2 1 2 3 2 2 2 3 2 3 2 1 2 3 2 1 1 3 2 1 3 2 1 1 2 2 3 1 1 1 3 1 1 2 2 3 2 3 2 3 1 1 2 2 3 1 2 3 1 3 2 2 2 3 1 1 2 2 2 3 2 2 2 3 1 3 2 1 1 2 3 1 2 3 2 1 2 1 1 2 3 1 2 3 2 3 2 3 2 1 1 1 2 2 1 2 3 2 3 1 3 1 3 1 1 3 1 1 2 2 2 3 2 2 2 1 2 2 3 2 3 1 2 1 1 1 3 2 1 2 2 3 2 2 3 1 2 1 3 1 1 1 3 1 1 3 2 1 3 1 1 2 1 3 1 1 1 3 2 2 1 1 2 1 3 1 2 2 3 2 3 2 1 3 2 2 1 1 3 1 3 2 2 3 2 2 2 1 1 2 2 1 3 2 1 3 2 1 1 3 2 2 3 2 2 1 3 1 1 2 1 3 2 2 1 1 2 2 2 3 1 1 3 2 1 2 1 1 2 3 1 1 2 3 2 3 2 3 2 1 3 1 1 1 2 2 3 2 1 3 2 1 2 2 2 3 1 3 1 3 1 1 2 3 2 1 2 1 2 3 2 2 1 1 2 3 1 3 1 2 3 2 2 3 2 1 2 1 2 2 2 3 1 2 1 1 3 1 3 1 1 2 3 1 1 3 1 1 3 2 2 2 3 1 1 2 1 3 2 3 2 1 1 2 3 1 1 2 1 2 3 1 2 3 3 2 1 3 2 2 2 3 2 3 1 1 2 1 3 1 1 2 2 1 3 2 2 2 1 1 1 3 1 2 3 1 2 2 3 2 1 1 2 2 2 3 2 3 2 3 1 1 3 1 1 3 1 2 2 3 2 2 3 1 3 2 2 1 1 2 1 3 1 2 1 1 1 3 1 2 2 1 2 3 2 1 3 2 3 1 2 3 2 1 1 1 2 3 2 2 3 1 1 2 2 2 1 3 1 2 3 2 1 3 1 2 1 2 3 1 1 2 3 2 3 1 2 1 3 1 1 3 2 3 2 1 2 2 1 1 3 2 1 1 3 2 2 1 2 1 2 3 1 1 2 2 1 2 3 1 3 1 1 3 1 1 2 1 3 1 3 2 2 2 2 3 2 2 1 2 3 1 1 3 2 3 1 2 2 2 3 2 2 2 3 2 3 2 1 1 1 3 1 2 2 3 2 3 2 2 1 2 1 2 3 1 1 1 2 3 2 2 3 2 3 1 2 1 3 2 1 3 2 2 1 3 1 2 1 2 2 2 3 2 3 1 1 2 2 1 1 3 1 2 1 1 1 3 1 1 3 1 3 1 1 3 2 1 3 1 2 2 3 2 1 3 1 1 2 3 1 1 2 2 2 3 2 1 3 2 1 2 1 1 1 2 1 1 3 1 3 1 3 1 3 1 1 2 3 1 2 2 2 1 3 2 1 1 2 2 1 2 3 2 3 1 1 2 1 3 1 2 2 3 2 2 3 1 1 3 2 2 1 1 3 1 2 2 2 1 2 3 2 3 1 2 1 3 2 1 3 1 3 2 2 2 1 1 1 3 1 2 1 3 2 3 2 2 2 3 2 2 3 2 3 2 2 1 2 1 2 2 3 1 2 2 2 1 2 3 1 1 3 1 3 2 1 2 1 3 2 3 1 1 1 2 2 2 3 1 2 3 1 3 2 1 3 2 2 2 1 1 3 1 3 1 1 2 1 1 1 3 2 2 3 2 2 2 3 1 2 3 2 2 2 3 1 1 2 3 3 1 2 2 3 2 3 1 2 3 1 1 2 1 1 2 3 2 2 1 2 2 3 1 3 1 2 3 1 1 3 1 1 1 2 1 2 3 1 2 1 2 3 1 1 2 1 3 2 2 1 1 1 3 2 2 1 2 2 3 1 1 3 2 3 1 1 3 2 2 3 1 2 2 3 2 1 1 3 1 1 1 2 1 3 1 3 1 2 2 2 3 2 3 2 2 3 1 3 1 2 2 3 1 3 2 2 2 1 1 3 2 1 2 2 1 3 1 2 2 1 3 2 3 1 2 1 1 2 1 3 1 1 2 3 1 2 1 1 1 2 3 2 3 3 1 2 1 1 2 1 3 2 3 1 1 2 2 2 3 1 3 2 2 3 2 1 2 1 3 1 2 1 2 2 2 3 2 1 3 2 1 3 1 1 1 3 2 1 2 3 2 3 2 2 1 2 3 1 1 2 3 2 2 3 1 1 2 2 2 3 1 1 2 3 2 1 2 3 1 1 1 3 1 2 2 2 1 3 2 2 3 2 3 1 3 1 2 1 2 1 1 1 2 1 3 1 3 1 1 3 2 2 1 2 3 1 2 3 2 3 1 2 1 2 2 1 3 2 3 1 3 1 1 1 2 3 2 2 2 1 1 2 3 2 3 1 2 2 3 1 1 3 1 1 2 1 2 3 2 3 1 1 1 2 2 1 3 2 2 2 3 3 2 2 2 3 1 2 1 3 2 2 2 1 1 2 3 1 3 2 1 2 2 3 1 3 2 2 3 2 1 1 3 2 1 1 2 3 1 2 1 1 1 3 2 1 2 3 1 2 1 1 3 1 3 2 1 3 2 1 1 2 2 3 2 2 3 2 2 2 1 3 1 2 2 2 3 1 3 1 3 1 3 2 1 2 3 2 1 2 3 1 2 2 1 2 2 1 2 2 3 1 2 2 3 2 3 1 1 2 2 1 3 1 2 1 3 1 1 3 1 3 1 2 2 1 3 2 1 2 2 2 1 3 2 1 3 2 1 1 2 1 3 1 3 2 1 2 3 2 1 2 2 1 3 1 3 1 2 1 2 2 3 1 1 1 3 2 3 2 1 2 3 2 3 1 1 1 3 2 1 1 2 3 1 2 1 1 1 2 3 1 3 3 2 1 1 2 2 1 3 2 1 1 2 3 1 2 2 2 3 1 1 2 3 1 3 3 2 2 2 1 2 2 3 2 1 1 1 3 1 2 3 2 1 1 3 2 3 1 1 2 1 3 2 1 3 1 1 2 2 3 2 2 3 2 2 1 1 1 3 1 1 2 3 2 1 2 2 3 2 2 1 3 2 2 1 2 3 2 1 3 2 3 2 3 2 1 1 3 1 3 2 3 1 1 1 3 2 2 1 2 1 2 3 1 1 1 3 2 1 2 1 1 2 1 2 1 3 1 1 3 2 2 3 1 2 3 1 3 2 2 2 1 2 3 1 2 2 2 1 3 1 1 3 2 1 1 3 1 1 2 1 1 3 2 3 1 3 2 1 2 3 2 3 2 1 2 1 1 3 1 2 1 2 2 2 3 2 1 1 3 1 1 3 2 1 3 1 1 3 1 3 2 2 3 2 1 2 2 3 2 2 1 2 1 1 3 2 3 2 3 2 2 1 2 2 1 3 2 2 2 1 1 3 2 2 1 3 1 3 2 1 1 1 2 1 2 1 3 2 3 1 2 3 2 3 1 1 1 2 2 3 1 1 2 3 2 2 1 3 1 3 1 1 2 1 3 1 3 2 3 1 2 2 1 2 1 3 2 2 3 1 1 3 2 3 1 3 2 2 1 1 2 3 1 2 2 2 3 2 1 1 1 2 1 1 2 3 2 1 1 1 3 2 1 1 1 3 1 1 1 3 2 3 1 2 3 1 3 2 2 1 3 2 2 1 2 3 1 2 3 1 1 2 1 2 2 3 2 3 2 1 1 1 1 2 3 1 3 2 2 1 3 1 3 2 1 3 1 1 2 2 1 2 3 2 3 1 2 1 2 1 3 1 1 3 1 2 2 1 3 2 2 1 3 2 3 1 2 1 1 2 1 3 2 2 2 3 2 2 3 1 3 1 2 2 2 1 2 3 1 3 2 1 2 1 3 1 1 2 1 3 2 2 1 3 2 1 3 2 1 1 3 1 3 2 1 2 3 1 1 2 2 2 3 2 1 2 2 3 2 3 1 1 3 2 2 2 1 3 2 1 3 2 1 3 2 1 1 3 1 1 3 1 3 1 1 2 2 1 3 1 2 2 1 1 1 1 2 3 2 3 2 2 1 2 3 2 1 2 3 2 1 1 1 2 1 3 2 3 3 1 1 2 2 1 3 2 2 1 3 1 3 2 1 1 1 2 2 3 2 2 2 3 3 1 1 1 2 2 3 1 1 3 1 2 1 3 2 1 1 3 1 1 1 2 3 1 3 2 3 2 1 2 2 1 2 3 2 3 1 2 2 2 1 2 3 1 2 1 3 1 2 1 2 2 1 2 3 1 3 1 1 1 3 2 2 3 1 1 2 1 3 2 1 3 2 1 2 3 2 1 2 2 3 2 1 2 2 3 1 3 2 1 3 1 2 3 1 1 3 2 3 1 2 2 3 1 1 2 1 3 2 1 3 1 2 2 3 2 2 2 1 1 1 3 2 1 1 3 2 2 3 2 2 2 3 1 2 2 3 1 1 1 2 2 2 3 3 1 1 3 2 2 2 3 1 2 2 2 1 1 3 2 2 2 1 1 3 1 1 3 3 1 3 1 1 3 1 2 1 1 1 2 3 1 2 1 2 2 3 2 2 1 2 3 1 2 3 1 2 3 1 3 2 2 3 2 2 1 1 2 1 3 2 2 1 3 2 2 2 1 2 3 1 2 1 2 2 2 3 1 1 3 1 3 2 3 2 2 1 1 3 1 3 1 3 1 2 3 1 2 2 1 1 1 3 2 3 1 2 2 2 1 2 3 1 1 1 2 1 3 2 2 1 1 3 1 3 2 3 1 2 3 1 3 1 1 2 1 1 1 2 2 2 1 2 2 3 2 2 1 3 1 2 1 1 1 3 1 3 2 2 3 1 3 1 3 1 1 2 1 2 2 3 1 2 1 3 2 2 3 1 1 3 2 2 3 1 1 2 1 3 2 3 2 1 1 1 3 2 3 2 1 3 1 2 2 3 2 1 1 1 2 1 3 2 1 3 2 3 1 2 1 2 3 1 2 2 2 3 1 1 2 1 2 2 3 2 3 2 1 2 2 3 1 1 2 2 1 3 1 1 2 1 3 2 3 1 3 1 1 2 3 1 2 1 2 3 1 3 1 2 1 3 1 1 3 2 2 2 1 1 2 3 2 3 1 1 3 1 1 3 2 1 1 3 2 1 2 1 1 1 3 2 1 1 1 2 3 2 2 2 1 1 3 2 3 2 3 1 2 1 1 3 1 1 1 3 1 2 1 3 1 2 1 2 2 3 2 2 3 1 1 2 3 2 3 2 2 2 1 1 1 3 1 3 1 3 1 1 2 1 1 2 3 1 2 3 1 3 1 2 3 1 2 2 1 2 2 3 1 2 1 3 1 3 1 1 1 3 1 3 1 3 1 1 2 2 3 2 1 2 2 1 1 1 2 3 2 1 2 1 1 2 3 1 3 1 2 1 2 3 2 2 2 3 2 3 1 1 1 2 1 3 1 2 1 1 3 1 2 2 3 1 2 2 3 2 3 2 2 2 3 2 2 2 3 1 2 3 1 2 1 1 2 1 3 1 1 3 1 3 1 1 2 3 1 1 3 1 2 3 1 1 2 1 1 3 2 2 3 2 3 1 1 2 3 2 2 2 1 1 3 1 2 3 1 1 1 3 1 1 1 3 2 3 2 1 3 1 1 2 1 2 2 2 3 2 2 1 1 1 2 3 2 1 2 3 2 1 3 2 1 1 2 2 3 1 3 2 1 3 2 1 3 2 3 2 3 1 1 3 2 2 1 2 2 2 3 2 2 1 2 1 3 2 3 1 1 2 3 2 2 2 3 2 1 1 1 3 1 3 2 2 2 1 1 3 1 2 1 1 1 2 3 1 3 1 1 2 2 3 1 3 2 1 1 2 2 3 2 2 3 1 2 3 1 3 1 1 1 2 2 3 2 2 2 1 1 3 2 3 2 2 2 1 1 1 2 1 1 3 2 1 3 2 3 2 3 1 3 2 1 1 2 1 3 2 1 2 1 2 3 1 1 1 2 1 2 3 2 3 1 2 1 3 2 1 1 3 1 3 1 1 2 2 3 2 1 1 3 1 3 2 3 1 2 2 1 2 1 3 1 2 3 1 2 1 3 1 3 2 1 1 3 1 1 2 3 1 1 1 3 1 3 1 2 1 1 2 1 2 1 1 3 2 1 1 3 2 1 3 1 2 3 2 2 1 1 1 3 1 3 1 2 1 1 1 2 1 3 1 1 1 3 1 1 2 2 3 2 1 3 1 3 2 1 3 2 1 2 1 3 1 2 2 2 1 1 3 2 3 1 1 3 1 3 1 3 2 2 1 2 3 1 1 2 3 2 2 2 3 2 1 1 1 2 3 2 1 2 1 3 1 2 1 3 1 1 1 2 1 3 1 1 2 3 1 3 2 1 3 2 3 1 1 1 2 1 2 3 2 2 3 1 1 2 2 1 2 3 2 1 3 1 3 1 1 1 3 2 1 1 1 3 2 1 3 2 1 1 1 2 2 3 1 3 1 3 2 1 3 2 2 3 1 1 2 2 2 3 2 1 1 1 3 2 3 2 2 2 1 2 1 3 2 3 2 3 2 1 1 2 1 2 1 2 3 1 2 2 2 3 1 3 1 2 3 1 3 1 1 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1 1 1 3 2 1 1 1 3 1 1 2 2 1 3 2 1 2 3 1 2 1 3 1 2 3 1 3 1 2 1 3 1 3 2 2 3 2 1 2 1 3 2 2 2 1 2 1 3 2 2 3 1 3 2 1 3 1 1 2 3 1 2 2 3 2 2 2 1 3 1 1 3 1 2 2 2 3 2 2 2 1 2 3 2 2 2 3 1 3 1 1 3 1 3 2 2 1 2 2 2 2 1 3 1 1 3 2 2 2 3 1 1 1 3 2 2 1 2 2 3 1 2 2 3 3 1 2 3 1 1 3 1 3 2 1 2 2 2 3 2 2 1 2 1 2 3 2 1 3 1 2 3 1 1 2 1 2 1 3 2 1 1 3 2 1 2 2 3 1 3 2 1 2 1 3 2 3 1 2 3 1 1 1 2 2 2 3 1 3 1 2 1 3 1 2 1 3 1 1 1 3 1 1 1 2 2 3 1 1 3 1 3 2 2 2 3 1 2 1 2 1 2 2 2 3 1 3 2 1 2 2 2 3 2 3 2 1 2 2 3 1 1 2 3 1 2 3 1 3 2 2 3 1 1 1 2 2 2 3 1 1 3 2 1 2 2 3 2 2 2 1 1 2 1 3 2 3 1 3 1 3 1 3 2 1 2 1 2 3 2 1 1 1 2 2 1 1 3 1 3 1 3 2 3 1 3 2 1 1 1 2 3 2 1 1 1 1 1 3 1 1 2 1 3 1 2 3 1 3 1 2 2 1 3 1 1 1 2 1 3 1 3 2 2 2 1 1 1 3 1 3 2 2 1 3 1 1 2 2 3 1 1 1 3 3 2 1 1 3 1 2 2 2 3 2 2 3 1 1 2 1 1 1 3 1 1 3 1 1 3 1 3 1 1 1 3 1 1 3 2 2 1 1 1 3 2 3 1 2 1 2 2 2 1 1 2 1 3 1 3 1 1 3 1 3 1 2 3 2 1 2 3 1 1 2 1 2 2 1 2 2 1 3 2 3 1 2 1 1 3 2 3 1 1 3 2 2 2 1 3 1 2 1 1 2 3 2 1 1 1 3 1 2 3 1 3 2 2 2 1 2 3 1 3 2 2 3 1 2 2 2 3 1 3 1 3 2 2 3 1 2 1 1 3 1 2 2 2 1 2 3 1 2 2 1 2 2 3 2 3 2 3 2 1 3 1 1 2 2 1 3 1 2 1 2 1 1 1 3 1 2 1 2 1 3 2 1 3 1 2 3 1 2 3 2 3 2 2 2 1 3 2 2 3 1 3 1 2 3 1 1 3 2 2 1 2 2 1 3 1 1 2 2 3 1 1 2 2 3 1 2 1 2 1 3 2 3 2 1 1 1 3 2 3 3 1 1 3 1 1 1 3 1 2 2 1 2 2 3 2 1 2 2 3 1 3 2 2 1 2 2 3 1 3 2 3 2 1 3 2 3 1 2 2 2 1 3 1 1 1 2 1 1 1 2 1 1 1 3 2 3 2 2 2 1 1 3 1 3 2 1 3 1 3 2 1 3 2 1 3 1 3 1 2 1 1 2 2 3 1 2 3 2 3 2 1 1 2 2 2

wherein each of 1 to 3 is a nucleotide base selected to be different from the others of 1 to 3 with the proviso that up to three nucleotide bases of each sequence can be substituted with any nucleotide base provided that: for any pair of sequences of the set: M1≦16, M2≦13, M3≦20, M4≦16, and M5≦19, where: M1 is the maximum number of matches for any alignment in which there are no internal indels; M2 is the maximum length of a block of matches for any alignment; M3 is the maximum number of matches for any alignment having a maximum score; M4 is the maximum sum of the lengths of the longest two blocks of matches for any alignment of maximum score; and M5 is the maximum sum of the lengths of all the blocks of matches having a length of at least 3, for any alignment of maximum score; wherein: the score of an alignment is determined according to the equation (A×m)−(B×mm)−(C×(og+eg))−(D×eg)), wherein: for each of (i) to (iv):  (i) m=6, mm=6, og=0 and eg=6,  (ii) m=6, mm=6, og=5 and eg=1,  (iii) m=6, mm=2, og=5 and eg=1, and  (iv) m=6, mm=6, og=6 and eg=0, A is the total number of matched pairs of bases in the alignment; B is the total number of internal mismatched pairs in the alignment; C is the total number of internal gaps in the alignment; and D is the total number of internal indels in the alignment minus the total number of internal gaps in the alignment; and wherein the maximum score is determined separately for each of (i), (ii)., (iii) and (iv). b) mixing said cleavage means, said target nucleic acid, said first and second oligonucleotides to create a reaction mixture under reaction conditions such that at least said 5′ portion of said first oligonucleotide is annealed to said target nucleic acid and wherein at least said 5′ and central portion of said second oligonucleotide is annealed to said target nucleic acid so as to create a cleavage structure and wherein the combined melting temperature of said complementary regions within said 5′ and 3′ portions of said first oligonucleotide when annealed to said target nucleic acid is greater than the melting temperature of said 5′ and central portion of said first oligonucleotide, and wherein cleavage of said cleavage structure occurs to generate non-target cleavage products; and c) detecting said non-target cleavage products.
 40. The composition of claim 39, wherein one or more of said first and second oligonucleotides contain a 3′-terminal dideoxynucleotide.
 41. The composition of claim 39, wherein the composition includes a plurality of said target nucleic acid sequences and a plurality of said second oligonucleotide molecules such that each of said second oligonucleotide molecules has a distinct 3′ region.
 43. The composition of claim 39, wherein the composition includes at least ten, or twenty, or thirty, or forty, or fifty, or sixty, or seventy, or eighty, or ninety, or one hundred, or one hundred and ten, or one hundred and twenty, or one hundred and thirty, or one hundred and forty, or one hundred and fity, or one hundred and sixty said second oligonucelotide molecules, or comprising one hundred and seventy said second oligonucleotide molecules, or comprising one hundred and eighty said second oligonucleotide molecules, or comprising one hundred and ninety said second oligonucleotide molecules, or comprising two hundred said second oligonucleotide molecules, or comprising two hundred and twenty said second oligonucleotide molecules, or comprising two hundred and forty said second oligonucleotide molecules, or comprising two hundred and sixty said second oligonucleotide molecules, or comprising two hundred and eighty said second oligonucleotide molecules, or comprising three hundred said second oligonucleotide molecules, or comprising four hundred said second oligonucleotide molecules, or comprising five hundred said second oligonucleotide molecules, or comprising six hundred said second oligonucleotide molecules, or comprising seven hundred said second oligonucleotide molecules, or comprising eight hundred said second oligonucleotide molecules, or comprising nine hundred said second oligonucleotide molecules, or comprising one thousand said second oligonucleotide molecules, or comprising eleven hundred said second oligonucleotide molecules.
 44. A method of analyzing a biological sample comprising a plurality of target nucleic acid molecules for the presence of a mutation or polymorphism at a locus of each target nucleic acid molecule, the method comprising: a) providing: i) a cleavage means, ii) a plurality of target nucleic acid molecules, each of said target nucleic acid molecules having a first region, a second region and a third region, wherein said first region is located adjacent to and downstream from said second region, and said second region is located adjacent to and downstream from said third region; iii) a plurality of first oligonucleotide molecules, each having a 5′ and a 3′ portion, said 5′ portion of said first oligonucleotide molecules having a sequence complementary to said second region of said target nucleic acid molecules and said 3′ portion of said first oligonucleotide molecules having a sequence complementary to said third region of said target nucleic acid molecules; iv) a plurality of second oligonucleotide molecules, each having a 5′ portion, a central portion and a 3′ portion, said 5′ portion of said second oligonucleotide molecules having a sequence complementary to said first region of said target nucleic acid molecules, said central portion of said second oligonucleotide molecules having a sequence complimentary to said second region of said target nucleic acid molecules, and said 3′ portion of said second oligonucleotide molecules having a sequence that is not base-paired to said target nucleic acid and is selected from a set of oligonucleotides based on a following group of sequences, 1 1 1 2 2 3 2 3 1 1 1 3 1 2 2 3 2 2 2 3 2 3 2 1 3 2 2 1 3 1 3 2 2 1 1 2 2 3 2 1 2 2 2 3 1 2 3 1 1 2 3 2 2 1 1 1 3 2 1 1 3 2 3 2 2 3 1 1 1 2 3 2 2 3 1 2 3 2 2 1 3 1 1 3 2 1 2 1 2 2 3 2 3 1 1 2 2 2 2 3 2 3 2 1 3 1 1 2 1 2 3 2 3 2 2 3 2 2 1 1 1 2 1 1 3 2 3 2 1 1 3 2 3 1 1 1 2 1 1 3 1 1 3 1 1 1 3 1 3 2 1 2 2 2 3 2 2 3 2 3 1 3 2 2 1 1 1 2 3 2 3 2 2 2 1 2 3 2 2 1 2 1 2 3 2 3 1 1 3 2 2 2 1 1 1 3 1 3 1 1 2 1 3 1 1 2 1 2 3 2 3 2 1 1 3 2 2 1 2 3 1 1 1 3 1 3 2 3 1 3 1 2 1 1 2 3 2 2 2 1 1 2 3 1 3 1 1 1 2 1 2 3 2 2 1 3 1 1 2 3 2 3 1 2 2 2 1 3 2 2 3 2 2 3 1 2 3 2 2 2 1 3 2 1 3 2 2 2 3 2 1 1 1 3 1 3 2 1 2 1 1 3 2 2 2 3 1 2 3 1 2 1 1 1 1 3 2 1 1 3 1 1 2 3 1 2 3 2 1 1 2 1 1 3 2 3 3 2 1 3 1 1 1 2 1 3 2 2 2 1 2 2 3 1 2 3 1 2 2 3 2 3 2 1 1 3 2 3 1 1 1 2 1 3 2 3 1 3 2 2 1 2 2 2 1 1 1 2 1 3 1 2 3 1 2 1 2 1 1 3 2 3 1 3 1 1 2 3 1 2 1 1 3 2 2 1 2 1 1 3 2 3 2 2 1 2 3 2 3 1 3 2 2 1 2 1 3 1 2 1 1 1 3 1 3 1 2 3 1 2 2 2 3 2 2 3 1 3 1 3 2 2 3 1 3 1 1 2 3 2 1 2 1 3 2 1 2 2 1 2 1 1 3 2 1 3 2 2 2 3 2 1 1 3 1 1 2 3 1 2 2 3 2 1 2 2 1 2 3 1 1 1 2 2 3 1 3 2 3 1 1 3 1 2 2 3 1 2 3 2 1 2 1 2 3 2 1 1 1 2 2 3 2 2 1 2 3 2 2 3 1 3 3 1 1 2 2 3 2 1 2 1 1 1 3 2 1 2 2 1 3 1 2 3 2 3 2 1 3 1 2 3 1 3 1 2 2 1 1 3 2 3 2 2 1 2 2 2 3 1 3 2 2 1 1 3 2 2 2 3 2 2 2 1 2 3 2 1 2 1 3 1 1 3 3 1 3 2 1 2 2 1 3 2 1 1 1 3 2 3 1 2 1 2 3 1 2 1 3 2 3 1 1 2 3 1 2 2 2 1 3 2 1 1 1 2 3 1 2 2 3 1 3 1 2 2 3 1 1 3 2 2 1 2 1 3 1 1 1 2 3 1 2 2 1 3 1 3 2 3 1 2 1 1 1 2 3 2 2 1 3 2 2 3 1 1 2 2 3 2 2 1 2 1 2 1 3 2 1 1 1 2 3 2 2 2 3 2 3 2 3 2 2 3 2 2 1 1 3 2 3 2 2 1 3 2 2 1 2 2 2 3 2 2 3 2 1 3 3 2 1 3 2 1 1 2 1 2 3 1 1 3 2 3 1 3 1 1 2 1 2 1 2 1 3 2 3 2 1 2 1 3 1 1 2 3 2 1 3 1 2 2 2 1 3 2 2 2 3 2 1 3 1 2 2 1 3 1 2 3 2 3 2 2 2 3 2 1 1 1 2 1 3 2 1 2 1 3 1 3 2 1 3 1 3 1 2 3 1 2 1 2 2 2 1 2 2 3 2 3 1 1 1 3 1 1 1 3 1 3 1 1 3 1 1 1 2 2 2 3 2 3 1 3 1 1 2 2 1 1 3 1 2 2 1 1 3 1 1 2 3 2 1 2 1 2 2 1 3 2 2 1 1 3 1 1 1 3 1 1 3 1 3 2 2 3 2 2 3 2 1 3 2 2 3 1 3 1 1 1 2 1 2 3 2 1 3 2 2 2 2 1 3 1 3 2 2 3 2 2 1 1 1 3 1 3 2 3 2 1 1 1 2 1 3 2 2 1 2 3 1 2 3 2 3 2 1 2 1 1 3 2 1 1 2 1 2 3 2 2 2 3 2 2 1 3 1 1 2 3 1 3 1 1 3 1 2 2 2 1 2 3 1 3 2 1 2 1 3 2 2 2 1 1 1 3 1 1 3 2 1 3 2 1 3 1 3 2 3 1 3 1 2 1 2 1 3 1 2 2 2 1 3 1 1 1 3 2 1 1 2 2 3 2 2 2 1 2 1 3 2 3 1 1 3 2 3 1 1 2 1 3 2 1 1 1 3 2 1 1 3 2 1 3 2 1 1 2 1 3 2 3 2 3 2 2 1 1 1 2 2 2 3 2 3 1 3 2 2 1 2 3 1 1 1 3 1 2 1 1 3 1 3 1 1 1 3 2 1 3 1 3 1 1 2 1 1 1 3 1 2 1 1 3 1 1 1 2 2 2 1 1 3 1 2 2 3 2 2 1 1 3 1 3 2 1 3 1 1 3 3 2 2 2 1 1 1 3 1 2 2 3 2 1 1 3 1 1 2 3 2 3 2 1 2 2 2 3 2 3 1 1 3 1 2 3 1 1 3 2 1 2 2 2 3 2 1 2 2 3 2 3 2 2 2 1 3 1 1 2 2 2 1 3 2 1 2 3 2 3 2 1 3 1 2 1 1 2 3 1 2 2 1 2 1 3 1 1 1 3 2 3 2 2 2 3 3 2 2 1 2 2 2 3 2 1 1 3 2 2 1 1 3 1 2 1 3 2 1 3 1 3 2 2 2 1 2 2 3 1 1 1 3 1 3 2 2 2 3 1 1 2 1 3 2 2 3 2 3 2 2 2 1 2 2 3 2 3 2 1 3 2 2 2 1 1 1 3 1 2 2 3 2 3 1 3 1 1 3 1 2 1 2 3 1 1 1 3 2 2 1 2 2 3 1 3 1 1 2 3 2 1 1 1 3 1 1 2 3 2 2 2 1 2 2 3 1 2 3 2 3 1 1 1 3 2 2 1 2 3 1 2 3 2 2 1 1 2 2 3 3 2 2 2 1 3 2 1 2 2 1 3 2 2 3 2 2 1 1 3 1 2 2 3 3 1 2 2 3 1 2 1 2 2 2 3 1 1 2 3 2 2 2 3 2 2 2 3 2 3 1 1 2 2 3 1 1 1 3 2 3 2 1 1 2 3 2 2 3 2 1 2 3 1 2 2 3 2 1 2 2 3 2 2 3 1 3 1 1 2 1 3 1 1 2 1 1 1 1 2 2 2 3 1 3 1 2 2 2 3 2 3 1 2 1 3 1 3 2 1 3 2 1 1 2 2 1 3 1 2 2 2 3 2 2 2 3 2 2 3 2 2 3 2 3 2 2 2 3 2 1 2 2 3 2 2 1 3 2 3 1 1 2 1 2 1 3 2 1 2 3 2 1 3 2 1 3 2 1 3 1 2 3 2 2 2 1 2 3 1 1 2 2 3 2 2 2 1 1 1 3 1 2 3 1 2 2 3 1 1 3 1 1 1 2 3 2 3 2 3 1 2 1 1 2 3 1 2 3 2 2 1 2 2 2 3 2 3 2 1 1 2 1 3 2 2 3 2 3 1 3 1 1 2 2 2 3 2 1 1 2 2 1 3 1 2 1 3 1 2 3 2 1 1 3 1 3 1 1 1 2 2 3 2 3 1 1 1 1 3 1 2 2 1 1 3 1 3 1 1 3 2 2 1 1 2 1 3 1 3 2 1 3 1 1 3 2 1 1 1 2 2 3 2 3 1 1 2 3 1 1 1 3 1 1 1 1 1 2 3 2 1 1 3 1 1 1 3 1 1 3 1 2 2 3 2 2 3 2 1 2 2 2 3 1 2 2 2 1 2 3 2 3 2 2 1 2 3 2 2 3 1 3 2 3 2 1 2 2 3 1 3 1 1 1 2 2 2 3 1 1 3 1 1 2 3 1 1 3 1 1 2 2 3 2 1 2 3 1 1 1 2 3 1 1 2 2 3 2 1 1 3 2 1 2 2 3 2 1 3 1 1 3 2 1 1 1 3 2 2 1 3 1 1 3 2 2 2 2 1 2 3 2 1 1 2 3 1 2 1 1 3 2 3 2 1 3 2 2 3 1 2 1 2 1 3 2 2 3 1 1 1 2 2 3 2 3 1 2 1 3 2 3 2 1 2 1 1 3 1 1 1 2 2 1 3 1 3 1 3 2 2 3 2 1 1 1 3 3 1 1 2 2 3 2 3 1 1 1 2 3 2 3 1 2 2 3 1 2 1 2 1 1 1 1 2 1 1 3 2 1 3 2 2 2 1 1 2 3 1 3 1 3 1 1 3 3 1 2 2 1 1 1 3 1 1 3 2 1 1 3 2 3 1 1 2 3 2 2 2 2 1 2 3 2 3 2 3 2 2 3 2 2 2 1 3 2 3 2 2 1 2 2 1 3 1 3 2 2 1 2 1 2 3 2 1 3 2 2 1 3 1 3 2 2 1 2 1 3 1 1 1 3 1 1 1 3 1 1 3 2 3 2 2 1 1 3 2 2 1 1 1 2 1 3 2 1 2 2 1 3 2 1 1 3 2 1 2 3 2 3 1 2 2 3 2 2 2 3 2 3 2 3 1 2 2 3 1 1 2 1 2 2 3 2 3 1 1 1 2 1 2 3 2 3 1 1 1 3 1 3 2 2 1 1 3 2 3 1 2 2 1 1 1 3 1 2 2 3 1 1 2 3 1 2 2 3 1 3 1 2 1 2 3 2 1 1 1 1 1 3 1 2 3 1 2 1 3 2 2 1 1 3 2 3 2 1 1 3 2 2 1 2 1 3 2 2 3 2 2 1 2 2 3 1 3 1 1 2 2 2 1 3 1 1 3 2 2 2 1 2 1 3 2 3 1 1 2 2 1 2 3 1 3 2 3 1 1 1 3 3 1 2 1 3 1 2 2 2 1 3 1 1 2 3 1 1 2 2 1 1 3 2 3 2 2 2 3 1 1 3 1 1 3 1 3 1 2 2 2 3 1 1 1 2 2 3 1 1 2 3 1 1 2 1 1 3 1 3 2 2 3 1 2 1 1 1 2 3 2 3 1 2 3 2 2 2 1 2 3 2 1 3 2 3 2 1 3 1 2 2 3 1 1 2 2 2 2 2 1 1 3 2 3 1 3 2 2 1 2 1 3 1 1 3 2 1 3 2 1 3 1 2 2 2 1 2 3 2 3 2 2 2 3 1 1 3 2 2 1 1 3 1 2 2 1 3 2 2 1 3 1 3 1 1 1 3 2 3 1 2 1 1 1 3 2 2 1 3 2 1 1 2 3 1 2 1 1 2 3 1 1 3 2 3 2 1 2 1 2 1 3 1 1 2 3 1 1 3 2 3 2 2 1 3 2 1 2 1 3 1 2 1 3 2 1 2 1 1 1 2 2 3 1 3 2 2 2 3 2 2 2 3 1 2 2 3 2 1 3 2 1 1 2 3 1 1 3 1 1 2 1 1 3 2 1 2 3 1 3 2 3 2 2 1 1 1 2 3 2 1 1 2 1 3 2 3 2 2 3 2 2 1 3 2 2 1 3 1 3 1 3 2 2 1 3 2 3 1 1 1 2 3 2 2 3 2 2 1 1 1 2 3 1 1 1 2 1 3 1 1 1 2 3 2 1 2 2 3 2 2 2 3 2 3 1 1 3 2 2 1 2 1 1 3 2 2 2 3 2 3 1 3 1 1 2 2 1 1 3 3 1 3 2 2 2 1 2 1 3 2 2 1 3 1 1 2 1 2 3 2 2 3 2 1 3 1 3 2 2 1 2 2 1 3 1 1 3 1 1 3 1 2 2 2 1 1 3 3 1 3 2 2 1 1 2 3 1 1 1 2 1 1 3 2 1 2 2 2 3 2 3 1 2 3 1 2 3 1 1 2 1 3 2 2 3 1 1 3 2 1 2 1 2 1 3 1 2 1 3 1 2 1 2 3 1 3 1 2 3 1 1 1 3 2 2 1 3 2 1 2 1 2 3 2 1 1 1 3 1 1 1 3 2 3 1 1 1 3 1 1 3 1 1 2 3 1 1 2 3 2 1 3 1 1 1 2 3 1 1 2 3 2 2 3 1 1 1 1 1 2 2 3 1 1 2 1 3 2 3 2 3 2 3 1 3 2 2 2 1 1 2 1 3 1 2 1 2 2 3 2 2 2 3 1 2 2 1 1 2 3 1 1 3 1 3 1 1 1 3 2 2 3 2 1 1 1 3 2 2 3 1 1 3 1 2 1 1 1 3 3 2 2 1 1 3 1 3 1 2 2 1 2 3 1 3 1 2 3 2 1 2 2 1 1 3 1 1 3 1 2 1 2 1 1 3 1 1 3 1 2 2 3 1 1 2 2 3 3 2 1 3 1 1 1 2 2 2 3 1 1 2 2 3 1 2 3 2 3 1 1 1 1 1 3 1 3 2 1 3 1 2 2 3 1 2 1 1 3 2 1 2 1 2 3 1 2 3 1 2 1 2 1 3 2 1 3 2 3 1 1 3 1 1 1 2 1 1 3 2 1 3 1 2 1 1 2 3 1 2 3 1 3 1 1 1 2 3 1 1 3 1 2 1 1 2 3 2 3 1 1 1 3 2 1 2 2 2 3 2 3 1 2 1 2 1 3 2 1 1 2 1 1 3 1 3 1 1 2 2 3 1 2 1 2 3 1 1 3 1 2 3 2 1 1 3 2 3 2 1 2 2 2 1 3 2 1 3 1 1 2 3 1 1 3 2 2 1 2 3 2 2 1 3 1 2 2 2 3 2 2 3 1 3 1 2 2 3 1 2 1 3 2 2 2 3 2 1 2 3 1 1 3 1 3 1 2 1 3 2 1 2 2 2 3 1 3 1 1 1 2 3 2 2 1 2 3 2 1 2 2 2 1 3 2 1 3 2 2 1 2 3 2 3 1 3 1 1 2 3 2 3 2 2 2 3 1 2 2 2 1 1 3 2 1 2 3 2 2 2 3 2 2 2 1 2 1 3 1 1 2 3 2 1 2 3 3 1 3 2 1 2 1 2 1 3 1 1 3 1 1 1 3 1 1 1 2 2 2 3 1 2 3 1 3 2 3 1 1 3 2 1 1 1 2 3 2 1 3 2 2 1 2 2 2 2 1 1 3 1 1 3 2 3 1 3 2 2 1 2 2 3 2 3 1 2 1 2 1 2 3 1 1 1 2 3 1 3 1 1 2 1 2 2 3 2 2 3 2 2 2 3 3 1 2 2 1 1 2 3 1 2 2 1 2 3 2 3 1 1 2 2 3 1 2 3 3 1 1 1 2 3 2 2 1 1 1 3 1 2 1 2 3 1 1 1 3 2 1 3 2 1 2 2 3 2 2 3 1 2 2 2 3 1 2 1 2 2 1 3 2 3 2 3 2 2 2 1 2 3 2 2 2 3 2 3 2 1 2 3 2 1 1 3 2 1 3 2 1 1 2 2 3 1 1 1 3 1 1 2 2 3 2 3 2 3 1 1 2 2 3 1 2 3 1 3 2 2 2 3 1 1 2 2 2 3 2 2 2 3 1 3 2 1 1 2 3 1 2 3 2 1 2 1 1 2 3 1 2 3 2 3 2 3 2 1 1 1 2 2 1 2 3 2 3 1 3 1 3 1 1 3 1 1 2 2 2 3 2 2 2 1 2 2 3 2 3 1 2 1 1 1 3 2 1 2 2 3 2 2 3 1 2 1 3 1 1 1 3 1 1 3 2 1 3 1 1 2 1 3 1 1 1 3 2 2 1 1 2 1 3 1 2 2 3 2 3 2 1 3 2 2 1 1 3 1 3 2 2 3 2 2 2 1 1 2 2 1 3 2 1 3 2 1 1 3 2 2 3 2 2 1 3 1 1 2 1 3 2 2 1 1 2 2 2 3 1 1 3 2 1 2 1 1 2 3 1 1 2 3 2 3 2 3 2 1 3 1 1 1 2 2 3 2 1 3 2 1 2 2 2 3 1 3 1 3 1 1 2 3 2 1 2 1 2 3 2 2 1 1 2 3 1 3 1 2 3 2 2 3 2 1 2 1 2 2 2 3 1 2 1 1 3 1 3 1 1 2 3 1 1 3 1 1 3 2 2 2 3 1 1 2 1 3 2 3 2 1 1 2 3 1 1 2 1 2 3 1 2 3 3 2 1 3 2 2 2 3 2 3 1 1 2 1 3 1 1 2 2 1 3 2 2 2 1 1 1 3 1 2 3 1 2 2 3 2 1 1 2 2 2 3 2 3 2 3 1 1 3 1 1 3 1 2 2 3 2 2 3 1 3 2 2 1 1 2 1 3 1 2 1 1 1 3 1 2 2 1 2 3 2 1 3 2 3 1 2 3 2 1 1 1 2 3 2 2 3 1 1 2 2 2 1 3 1 2 3 2 1 3 1 2 1 2 3 1 1 2 3 2 3 1 2 1 3 1 1 3 2 3 2 1 2 2 1 1 3 2 1 1 3 2 2 1 2 1 2 3 1 1 2 2 1 2 3 1 3 1 1 3 1 1 2 1 3 1 3 2 2 2 2 3 2 2 1 2 3 1 1 3 2 3 1 2 2 2 3 2 2 2 3 2 3 2 1 1 1 3 1 2 2 3 2 3 2 2 1 2 1 2 3 1 1 1 2 3 2 2 3 2 3 1 2 1 3 2 1 3 2 2 1 3 1 2 1 2 2 2 3 2 3 1 1 2 2 1 1 3 1 2 1 1 1 3 1 1 3 1 3 1 1 3 2 1 3 1 2 2 3 2 1 3 1 1 2 3 1 1 2 2 2 3 2 1 3 2 1 2 1 1 1 2 1 1 3 1 3 1 3 1 3 1 1 2 3 1 2 2 2 1 3 2 1 1 2 2 1 2 3 2 3 1 1 2 1 3 1 2 2 3 2 2 3 1 1 3 2 2 1 1 3 1 2 2 2 1 2 3 2 3 1 2 1 3 2 1 3 1 3 2 2 2 1 1 1 3 1 2 1 3 2 3 2 2 2 3 2 2 3 2 3 2 2 1 2 1 2 2 3 1 2 2 2 1 2 3 1 1 3 1 3 2 1 2 1 3 2 3 1 1 1 2 2 2 3 1 2 3 1 3 2 1 3 2 2 2 1 1 3 1 3 1 1 2 1 1 1 3 2 2 3 2 2 2 3 1 2 3 2 2 2 3 1 1 2 3 3 1 2 2 3 2 3 1 2 3 1 1 2 1 1 2 3 2 2 1 2 2 3 1 3 1 2 3 1 1 3 1 1 1 2 1 2 3 1 2 1 2 3 1 1 2 1 3 2 2 1 1 1 3 2 2 1 2 2 3 1 1 3 2 3 1 1 3 2 2 3 1 2 2 3 2 1 1 3 1 1 1 2 1 3 1 3 1 2 2 2 3 2 3 2 2 3 1 3 1 2 2 3 1 3 2 2 2 1 1 3 2 1 2 2 1 3 1 2 2 1 3 2 3 1 2 1 1 2 1 3 1 1 2 3 1 2 1 1 1 2 3 2 3 3 1 2 1 1 2 1 3 2 3 1 1 2 2 2 3 1 3 2 2 3 2 1 2 1 3 1 2 1 2 2 2 3 2 1 3 2 1 3 1 1 1 3 2 1 2 3 2 3 2 2 1 2 3 1 1 2 3 2 2 3 1 1 2 2 2 3 1 1 2 3 2 1 2 3 1 1 1 3 1 2 2 2 1 3 2 2 3 2 3 1 3 1 2 1 2 1 1 1 2 1 3 1 3 1 1 3 2 2 1 2 3 1 2 3 2 3 1 2 1 2 2 1 3 2 3 1 3 1 1 1 2 3 2 2 2 1 1 2 3 2 3 1 2 2 3 1 1 3 1 1 2 1 2 3 2 3 1 1 1 2 2 1 3 2 2 2 3 3 2 2 2 3 1 2 1 3 2 2 2 1 1 2 3 1 3 2 1 2 2 3 1 3 2 2 3 2 1 1 3 2 1 1 2 3 1 2 1 1 1 3 2 1 2 3 1 2 1 1 3 1 3 2 1 3 2 1 1 2 2 3 2 2 3 2 2 2 1 3 1 2 2 2 3 1 3 1 3 1 3 2 1 2 3 2 1 2 3 1 2 2 1 2 2 1 2 2 3 1 2 2 3 2 3 1 1 2 2 1 3 1 2 1 3 1 1 3 1 3 1 2 2 1 3 2 1 2 2 2 1 3 2 1 3 2 1 1 2 1 3 1 3 2 1 2 3 2 1 2 2 1 3 1 3 1 2 1 2 2 3 1 1 1 3 2 3 2 1 2 3 2 3 1 1 1 3 2 1 1 2 3 1 2 1 1 1 2 3 1 3 3 2 1 1 2 2 1 3 2 1 1 2 3 1 2 2 2 3 1 1 2 3 1 3 3 2 2 2 1 2 2 3 2 1 1 1 3 1 2 3 2 1 1 3 2 3 1 1 2 1 3 2 1 3 1 1 2 2 3 2 2 3 2 2 1 1 1 3 1 1 2 3 2 1 2 2 3 2 2 1 3 2 2 1 2 3 2 1 3 2 3 2 3 2 1 1 3 1 3 2 3 1 1 1 3 2 2 1 2 1 2 3 1 1 1 3 2 1 2 1 1 2 1 2 1 3 1 1 3 2 2 3 1 2 3 1 3 2 2 2 1 2 3 1 2 2 2 1 3 1 1 3 2 1 1 3 1 1 2 1 1 3 2 3 1 3 2 1 2 3 2 3 2 1 2 1 1 3 1 2 1 2 2 2 3 2 1 1 3 1 1 3 2 1 3 1 1 3 1 3 2 2 3 2 1 2 2 3 2 2 1 2 1 1 3 2 3 2 3 2 2 1 2 2 1 3 2 2 2 1 1 3 2 2 1 3 1 3 2 1 1 1 2 1 2 1 3 2 3 1 2 3 2 3 1 1 1 2 2 3 1 1 2 3 2 2 1 3 1 3 1 1 2 1 3 1 3 2 3 1 2 2 1 2 1 3 2 2 3 1 1 3 2 3 1 3 2 2 1 1 2 3 1 2 2 2 3 2 1 1 1 2 1 1 2 3 2 1 1 1 3 2 1 1 1 3 1 1 1 3 2 3 1 2 3 1 3 2 2 1 3 2 2 1 2 3 1 2 3 1 1 2 1 2 2 3 2 3 2 1 1 1 1 2 3 1 3 2 2 1 3 1 3 2 1 3 1 1 2 2 1 2 3 2 3 1 2 1 2 1 3 1 1 3 1 2 2 1 3 2 2 1 3 2 3 1 2 1 1 2 1 3 2 2 2 3 2 2 3 1 3 1 2 2 2 1 2 3 1 3 2 1 2 1 3 1 1 2 1 3 2 2 1 3 2 1 3 2 1 1 3 1 3 2 1 2 3 1 1 2 2 2 3 2 1 2 2 3 2 3 1 1 3 2 2 2 1 3 2 1 3 2 1 3 2 1 1 3 1 1 3 1 3 1 1 2 2 1 3 1 2 2 1 1 1 1 2 3 2 3 2 2 1 2 3 2 1 2 3 2 1 1 1 2 1 3 2 3 3 1 1 2 2 1 3 2 2 1 3 1 3 2 1 1 1 2 2 3 2 2 2 3 3 1 1 1 2 2 3 1 1 3 1 2 1 3 2 1 1 3 1 1 1 2 3 1 3 2 3 2 1 2 2 1 2 3 2 3 1 2 2 2 1 2 3 1 2 1 3 1 2 1 2 2 1 2 3 1 3 1 1 1 3 2 2 3 1 1 2 1 3 2 1 3 2 1 2 3 2 1 2 2 3 2 1 2 2 3 1 3 2 1 3 1 2 3 1 1 3 2 3 1 2 2 3 1 1 2 1 3 2 1 3 1 2 2 3 2 2 2 1 1 1 3 2 1 1 3 2 2 3 2 2 2 3 1 2 2 3 1 1 1 2 2 2 3 3 1 1 3 2 2 2 3 1 2 2 2 1 1 3 2 2 2 1 1 3 1 1 3 3 1 3 1 1 3 1 2 1 1 1 2 3 1 2 1 2 2 3 2 2 1 2 3 1 2 3 1 2 3 1 3 2 2 3 2 2 1 1 2 1 3 2 2 1 3 2 2 2 1 2 3 1 2 1 2 2 2 3 1 1 3 1 3 2 3 2 2 1 1 3 1 3 1 3 1 2 3 1 2 2 1 1 1 3 2 3 1 2 2 2 1 2 3 1 1 1 2 1 3 2 2 1 1 3 1 3 2 3 1 2 3 1 3 1 1 2 1 1 1 2 2 2 1 2 2 3 2 2 1 3 1 2 1 1 1 3 1 3 2 2 3 1 3 1 3 1 1 2 1 2 2 3 1 2 1 3 2 2 3 1 1 3 2 2 3 1 1 2 1 3 2 3 2 1 1 1 3 2 3 2 1 3 1 2 2 3 2 1 1 1 2 1 3 2 1 3 2 3 1 2 1 2 3 1 2 2 2 3 1 1 2 1 2 2 3 2 3 2 1 2 2 3 1 1 2 2 1 3 1 1 2 1 3 2 3 1 3 1 1 2 3 1 2 1 2 3 1 3 1 2 1 3 1 1 3 2 2 2 1 1 2 3 2 3 1 1 3 1 1 3 2 1 1 3 2 1 2 1 1 1 3 2 1 1 1 2 3 2 2 2 1 1 3 2 3 2 3 1 2 1 1 3 1 1 1 3 1 2 1 3 1 2 1 2 2 3 2 2 3 1 1 2 3 2 3 2 2 2 1 1 1 3 1 3 1 3 1 1 2 1 1 2 3 1 2 3 1 3 1 2 3 1 2 2 1 2 2 3 1 2 1 3 1 3 1 1 1 3 1 3 1 3 1 1 2 2 3 2 1 2 2 1 1 1 2 3 2 1 2 1 1 2 3 1 3 1 2 1 2 3 2 2 2 3 2 3 1 1 1 2 1 3 1 2 1 1 3 1 2 2 3 1 2 2 3 2 3 2 2 2 3 2 2 2 3 1 2 3 1 2 1 1 2 1 3 1 1 3 1 3 1 1 2 3 1 1 3 1 2 3 1 1 2 1 1 3 2 2 3 2 3 1 1 2 3 2 2 2 1 1 3 1 2 3 1 1 1 3 1 1 1 3 2 3 2 1 3 1 1 2 1 2 2 2 3 2 2 1 1 1 2 3 2 1 2 3 2 1 3 2 1 1 2 2 3 1 3 2 1 3 2 1 3 2 3 2 3 1 1 3 2 2 1 2 2 2 3 2 2 1 2 1 3 2 3 1 1 2 3 2 2 2 3 2 1 1 1 3 1 3 2 2 2 1 1 3 1 2 1 1 1 2 3 1 3 1 1 2 2 3 1 3 2 1 1 2 2 3 2 2 3 1 2 3 1 3 1 1 1 2 2 3 2 2 2 1 1 3 2 3 2 2 2 1 1 1 2 1 1 3 2 1 3 2 3 2 3 1 3 2 1 1 2 1 3 2 1 2 1 2 3 1 1 1 2 1 2 3 2 3 1 2 1 3 2 1 1 3 1 3 1 1 2 2 3 2 1 1 3 1 3 2 3 1 2 2 1 2 1 3 1 2 3 1 2 1 3 1 3 2 1 1 3 1 1 2 3 1 1 1 3 1 3 1 2 1 1 2 1 2 1 1 3 2 1 1 3 2 1 3 1 2 3 2 2 1 1 1 3 1 3 1 2 1 1 1 2 1 3 1 1 1 3 1 1 2 2 3 2 1 3 1 3 2 1 3 2 1 2 1 3 1 2 2 2 1 1 3 2 3 1 1 3 1 3 1 3 2 2 1 2 3 1 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2 1 1 3 2 2 2 3 1 3 2 2 2 3 1 1 1 3 1 3 2 3 1 1 2 1 3 1 1 1 2 1 1 3 1 3 1 1 1 1 2 1 1 1 3 2 2 1 2 2 3 1 3 1 3 1 3 2 2 2 1 3 1 3 2 1 3 2 3 2 2 3 2 1 3 2 2 2 1 3 2 1 2 1 2 1 3 2 1 1 3 1 1 2 3 2 1 2 2 1 3 1 2 1 2 2 2 3 2 3 3 1 2 1 1 1 2 3 2 2 2 3 1 2 1 1 1 3 2 1 3 2 2 3 1 2 1 3 2 1 2 3 2 1 2 3 2 3 2 3 1 1 3 1 2 2 2 1 1 2 3 1 1 2 3 2 1 3 1 3 2 3 1 2 2 1 3 2 2 2 1 1 3 2 1 3 2 1 2 2 2 1 3 2 3 1 2 3 2 1 1 3 1 1 2 1 1 3 1 1 2 2 3 2 1 2 2 3 1 1 3 1 1 3 1 1 2 1 2 3 2 2 2 1 2 1 3 1 1 2 2 3 1 3 1 3 1 1 3 2 2 1 1 3 1 1 1 3 2 1 3 2 1 3 1 3 1 2 2 2 3 1 3 1 1 2 2 1 2 2 2 1 1 1 3 1 1 1 3 2 1 2 2 3 2 1 1 3 1 3 2 3 1 1 1 2 2 3 1 3 1 1 1 3 2 3 1 1 2 3 1 1 3 2 2 2 1 1 3 1 1 1 2 1 1 3 2 1 2 3 1 2 1 3 2 1 3 2 1 3 1 2 2 2 3 1 1 2 2 3 2 1 2 2 3 2 1 3 2 2 2 3 2 3 1 1 3 1 3 1 1 2 1 1 2 3 2 1 3 1 3 1 2 1 2 1 1 3 2 3 2 3 2 1 1 2 1 3 2 2 3 2 2 1 1 2 3 1 3 2 1 1 2 1 2 1 3 2 2 3 2 1 3 2 2 2 1 3 1 2 3 1 1 2 3 2 1 2 2 3 2 3 2 2 1 3 1 1 2 3 1 2 3 2 2 1 1 2 1 3 3 2 2 2 3 2 1 2 1 3 2 1 2 2 2 3 1 2 2 3 1 2 3 2 1 3 1 3 2 1 1 1 3 2 1 2 3 1 3 2 2 1 2 3 1 1 2 1 3 1 1 1 3 2 2 2 1 1 3 2 3 1 2 3 2 1 2 1 2 2 3 2 2 2 1 1 1 2 3 1 2 1 1 1 3 1 3 2 1 3 2 3 1 1 3 2 2 2 1 1 1 2 3 2 3 2 3 1 3 1 1 3 1 2 3 1 1 2 1 1 1 2 2 2 3 2 1 2 1 1 1 3 2 3 1 1 3 1 1 3 1 3 1 1 2 3 1 2 2 1 3 2 1 2 2 2 3 2 3 1 1 3 1 3 1 2 2 2 2 2 2 3 1 1 2 3 1 1 1 2 2 3 1 2 3 1 2 1 3 1 2 3 1 3 1 3 2 1 1 3 1 2 2 1 1 3 1 1 2 1 1 3 1 1 1 3 1 2 2 3 1 1 2 2 3 1 3 1 1 3 2 3 1 1 3 2 1 1 1 2 2 2 2 1 3 1 3 1 1 3 2 1 2 2 3 2 2 2 3 1 1 1 3 1 2 1 1 1 3 2 3 1 1 1 3 1 2 2 2 3 1 1 1 2 3 1 2 3 3 1 1 1 3 2 2 1 3 1 3 1 1 1 2 3 2 1 3 1 1 1 2 2 3 2 3 1 1 2 1 1 2 3 1 1 3 1 1 3 2 2 1 2 3 2 2 1 2 2 3 2 3 1 1 2 1 1 1 3 2 1 3 1 2 3 2 3 2 2 1 2 2 2 1 2 1 2 3 1 2 1 2 3 1 3 2 2 2 3 2 3 2 2 3 1 2 2 3 1 2 2 2 3 2 3 2 3 1 3 2 1 2 2 1 3 2 2 1 2 1 1 1 3 2 3 1 2 2 1 1 3 2 2 1 3 2 2 2 3 1 3 1 2 2 2 3 2 1 2 2 2 3 2 1 2 1 1 2 3 2 2 3 1 1 3 1 3 3 2 2 2 3 1 1 1 2 2 1 3 2 3 2 3 1 3 1 1 1 2 1 2 1 1 2 3 2 2 3 1 3 1 2 2 3 1 2 1 1 2 3 2 2 3 1 1 2 1 3 2 1 3 2 1 3 2 1 2 2 3 2 2 3 2 1 1 2 1 1 3 3 2 2 3 2 1 1 2 2 2 3 1 3 2 3 2 2 1 3 2 2 1 2 2 2 3 1 1 2 1 2 3 1 2 1 3 2 2 1 3 2 1 1 2 2 3 2 3 2 3 1 2 1 3 2 1 2 3 2 2 2 3 2 3 1 2 2 1 1 1 3 1 3 1 2 3 2 1 2 1 1 1 3 1 3 2 1 2 3 2 2 1 2 1 1 3 1 3 2 3 1 3 1 2 2 2 1 3 1 1 3 1 2 3 2 2 1 2 2 1 1 2 3 1 3 1 1 2 2 2 3 2 2 1 1 1 3 1 3 1 1 1 3 2 1 1 1 3 1 1 2 2 1 3 2 1 2 3 1 2 1 3 1 2 3 1 3 1 2 1 3 1 3 2 2 3 2 1 2 1 3 2 2 2 1 2 1 3 2 2 3 1 3 2 1 3 1 1 2 3 1 2 2 3 2 2 2 1 3 1 1 3 1 2 2 2 3 2 2 2 1 2 3 2 2 2 3 1 3 1 1 3 1 3 2 2 1 2 2 2 2 1 3 1 1 3 2 2 2 3 1 1 1 3 2 2 1 2 2 3 1 2 2 3 3 1 2 3 1 1 3 1 3 2 1 2 2 2 3 2 2 1 2 1 2 3 2 1 3 1 2 3 1 1 2 1 2 1 3 2 1 1 3 2 1 2 2 3 1 3 2 1 2 1 3 2 3 1 2 3 1 1 1 2 2 2 3 1 3 1 2 1 3 1 2 1 3 1 1 1 3 1 1 1 2 2 3 1 1 3 1 3 2 2 2 3 1 2 1 2 1 2 2 2 3 1 3 2 1 2 2 2 3 2 3 2 1 2 2 3 1 1 2 3 1 2 3 1 3 2 2 3 1 1 1 2 2 2 3 1 1 3 2 1 2 2 3 2 2 2 1 1 2 1 3 2 3 1 3 1 3 1 3 2 1 2 1 2 3 2 1 1 1 2 2 1 1 3 1 3 1 3 2 3 1 3 2 1 1 1 2 3 2 1 1 1 1 1 3 1 1 2 1 3 1 2 3 1 3 1 2 2 1 3 1 1 1 2 1 3 1 3 2 2 2 1 1 1 3 1 3 2 2 1 3 1 1 2 2 3 1 1 1 3 3 2 1 1 3 1 2 2 2 3 2 2 3 1 1 2 1 1 1 3 1 1 3 1 1 3 1 3 1 1 1 3 1 1 3 2 2 1 1 1 3 2 3 1 2 1 2 2 2 1 1 2 1 3 1 3 1 1 3 1 3 1 2 3 2 1 2 3 1 1 2 1 2 2 1 2 2 1 3 2 3 1 2 1 1 3 2 3 1 1 3 2 2 2 1 3 1 2 1 1 2 3 2 1 1 1 3 1 2 3 1 3 2 2 2 1 2 3 1 3 2 2 3 1 2 2 2 3 1 3 1 3 2 2 3 1 2 1 1 3 1 2 2 2 1 2 3 1 2 2 1 2 2 3 2 3 2 3 2 1 3 1 1 2 2 1 3 1 2 1 2 1 1 1 3 1 2 1 2 1 3 2 1 3 1 2 3 1 2 3 2 3 2 2 2 1 3 2 2 3 1 3 1 2 3 1 1 3 2 2 1 2 2 1 3 1 1 2 2 3 1 1 2 2 3 1 2 1 2 1 3 2 3 2 1 1 1 3 2 3 3 1 1 3 1 1 1 3 1 2 2 1 2 2 3 2 1 2 2 3 1 3 2 2 1 2 2 3 1 3 2 3 2 1 3 2 3 1 2 2 2 1 3 1 1 1 2 1 1 1 2 1 1 1 3 2 3 2 2 2 1 1 3 1 3 2 1 3 1 3 2 1 3 2 1 3 1 3 1 2 1 1 2 2 3 1 2 3 2 3 2 1 1 2 2 2

wherein each of 1 to 3 is a nucleotide base selected to be different from the others of 1 to 3 with the proviso that up to three nucleotide bases of each sequence can be substituted with any nucleotide base provided that: for any pair of sequences of the set: M1≦16, M2≦13, M3≦20, M4≦16, and M5≦19, where: M1 is the maximum number of matches for any alignment in which there are no internal indels; M2 is the maximum length of a block of matches for any alignment; M3 is the maximum number of matches for any alignment having a maximum score; M4 is the maximum sum of the lengths of the longest two blocks of matches for any alignment of maximum score; and M5 is the maximum sum of the lengths of all the blocks of matches having a length of at least 3, for any alignment of maximum score; wherein: the score of an alignment is determined according to the equation (A×m)−(B×mm)−(C×(og+eg))−(D×eg)), wherein: for each of (i) to (iv):  (i) m=6, mm=6, og=0 and eg=6,  (ii) m=6, mm=6, og=5 and eg=1,  (iii) m=6, mm=0.2, og=5 and eg=1, and  (iv) m=6, mm=6, og=6 and eg=0, A is the total number of matched pairs of bases in the alignment; B is the total number of internal mismatched pairs in the alignment; C is the total number of internal gaps in the alignment; and D is the total number of internal indels in the alignment minus the total number of internal gaps in the alignment; and wherein the maximum score is determined separately for each of (i), (ii), (iii) and (iv). b) mixing said cleavage means, said target nucleic acid, said first and second oligonucleotides to create a reaction mixture under reaction conditions such that at least said 5′ portion of said first oligonucleotide is annealed to said target nucleic acid and wherein at least said 5′ and central portion of said second oligonucleotide is annealed to said target nucleic acid so as to create a cleavage structure and wherein the combined melting temperature of said complementary regions within said 5′ and 3′ portions of said first oligonucleotide when annealed to said target nucleic acid is greater than the melting temperature of said 5′ and central portion of said first oligonucleotide, and wherein cleavage of said cleavage structure occurs to generate non-target cleavage products; and c) detecting said non-target cleavage products.
 45. The method of claim 44, wherein said reaction temperature is between approximately 50 and 70 degrees centigrade.
 46. The method of claim 44; wherein said target nucleic acid molecules comprises single-stranded DNA.
 47. The method of claim 44, wherein said target nucleic acid molecules comprises double-stranded DNA and prior to step (c), said reaction mixture is treated such that said double-stranded DNA is rendered substantially single-stranded.
 48. The method of claim 47, wherein said treatment to render said double-stranded DNA is rendered substantially single-stranded by increasing the temperature.
 49. The method of claim 44, wherein said target nucleic acid molecules comprises RNA and wherein said first and second oligonucleotide molecules comprise DNA.
 50. The method of claim 44, wherein said cleavage means comprises a thermostable 5′ nuclease.
 51. The method of claim 50, wherein a portion of the amino acid sequence is homologous to a portion of the amino acid sequence of a thermostable DNA polymerase derived from a thermophilic organism.
 52. The method of claim 51, wherein said organism is selected from the group consisting of Thermus aquaticus, Thermus flavus and Thermus thermophilus.
 53. The method of claim 44, wherein said source of target nucleic acid molecules comprises a sample containing genomic DNA.
 54. The method of claim 44, wherein said reaction conditions comprise providing a source of divalent cations.
 55. A method of detecting the presence of a target nucleic acid molecule by detecting non-target cleavage products, the method comprising: a) providing: i) a cleavage means, ii) a first target nucleic acid, said first target nucleic acid having a first region, a second region and a third region, wherein said first region is located adjacent to and downstream from said second region, and said second region is located adjacent to and downstream from said third region; iii) a first oligonucleotide having a 5′ and a 3′ portion, said 5′ portion of said first oligonucleotide having a sequence complementary to said second region of said target nucleic acid and said 3′ portion of said first oligonucleotide having a sequence complementary to said third region of said target nucleic acid; iv) a second oligonucleotide having a 5′ portion, a central portion and a 3′ portion, said 5′ portion of said second oligonucleotide having a sequence complementary to said first region of said target nucleic acid, said central portion of said second oligonucleotide having a sequence complimentary to said second region of said target nucleic acid, and said 3′ portion of said second oligonucleotide having a sequence that is not base-paired to said target nucleic acid and is selected from a set of oligonucleotides, based on a following group of sequences each having a 3′ and 5′ portion, 1 4 6 6 1 3 2 4 5 5 2 3 1 8 1 2 3 4 1 7 1 9 8 4 1 1 9 2 6 9 1 2 4 3 9 6 9 8 9 8 10 9 9 1 2 3 8 10 8 8 7 4 3 1 1 1 1 1 1 2 2 1 3 3 2 2 3 1 2 2 3 2 4 1 4 4 4 2 1 2 3 3 1 1 1 3 2 2 1 4 3 3 3 3 3 4 4 3 1 1 4 4 3 4 1 1 3 3 3 6 6 6 3 5 6 6 1 1 6 5 7 6 7 7 7 5 8 7 5 5 8 8 2 1 7 7 1 1 2 3 2 3 1 3 2 6 5 6 1 6 4 8 1 1 3 8 5 3 1 1 6 3 5 6 8 8 6 6 8 3 6 5 7 3 1 2 3 1 4 6 1 5 7 5 4 3 2 1 6 7 3 6 2 6 1 3 3 1 2 7 6 8 3 1 3 4 3 1 2 5 3 5 6 1 2 7 3 6 1 7 2 7 4 6 3 5 1 7 5 4 6 3 8 6 6 8 2 3 7 1 7 1 7 8 6 3 7 3 4 1 6 8 4 7 7 1 2 4 3 6 5 2 6 3 1 4 1 4 6 1 3 3 1 4 8 1 8 3 3 5 3 8 1 3 6 6 3 7 7 3 8 6 4 7 3 1 3 7 8 6 10 9 5 5 10 10 7 10 10 10 7 9 9 9 7 7 10 9 9 3 10 3 10 3 9 6 3 4 10 6 10 4 10 3 9 4 3 9 3 10 4 9 9 10 5 9 4 8 3 9 4 9 10 7 3 5 9 4 10 8 4 10 5 4 9 3 5 3 3 9 8 10 6 8 6 9 7 10 4 6 10 9 6 4 4 9 8 10 8 3 7 7 9 10 5 3 8 8 9 3 9 10 8 10 2 9 5 9 9 6 2 2 7 10 9 7 5 3 10 6 10 3 6 8 9 2 10 9 3 2 7 3 8 9 10 3 6 2 3 2 5 10 8 9 8 2 3 10 2 9 6 3 9 8 2 10 3 7 3 9 9 10 9 10 1 1 9 4 10 1 9 1 4 1 7 1 10 9 8 1 9 1 10 1 10 6 9 6 9 1 3 10 3 10 8 8 9 1 3 8 1 9 10 3 9 10 1 3 6 9 1 9 1 10 3 1 1 4 9 6 8 10 3 3 9 6 1 10 5 3 1 6 9 10 6 1 8 10 9 6 5 9 9 4 10 3 2 10 9 1 9 5 10 10 7 2 1 9 10 9 9 1 8 2 1 8 6 8 9 10 1 9 1 3 8 10 9 6 9 10 1 2 1 10 8 9 9 2 1 9 6 7 2 9 4 3 9 3 5 1 5 11 10 14 12 1 7 12 4 13 3 2 5 5 4 4 12 9 2 13 13 11 13 13 10 2 5 4 12 7 11 7 4 11 6 4 12 12 1 9 11 11 12 9 4 14 12 6 12 7 13 2 9 11 9 11 3 4 1 3 10 5 12 11 4 4 4 13 7 12 1 5 9 13 10 11 11 6 10 14 14 10 1 3 2 14 1 10 4 5 10 12 12 7 11 10 9 11 2 12 8 11 2 8 5 2 12 14 1 8 13 3 7 8 9 4 7 5 4 2 13 2 12 7 1 12 11 10 9 7 5 11 8 12 2 2 12 7 5 2 14 3 4 13 1 8 8 1 5 9 14 5 11 10 13 3 14 1 4 13 2 4 4 4 5 11 3 10 10 9 2 3 3 11 11 4 8 14 3 4 5 1 14 8 11 2 14 3 11 6 12 5 13 4 4 1 10 1 6 10 11 6 5 1 5 8 12 5 1 7 4 5 9 6 9 2 13 2 4 4 2 3 11 2 2 5 9 3 8 1 10 12 2 8 12 7 9 11 4 1 12 1 4 14 3 13 11 2 7 10 4 1 3 4 12 11 11 11 3 3 4 2 12 11 1 5 9 4 2 1 6 1 12 2 10 5 10 5 1 12 2 14 2 11 7 9 4 11 7 4 4 5 14 12 12 5 2 1 10 12 5 9 2 11 6 1 12 14 3 6 1 14 5 9 11 10 1 4 2 5 12 14 10 10 4 5 8 4 5 6 10 12 4 6 12 5 4 2 1 13 6 8 9 10 10 14 5 3 6 14 10 11 3 3 2 9 10 12 5 7 13 3 7 10 5 12 6 4 1 2 5 13 6 1 13 4 14 13 2 12 1 14 1 9 4 11 13 2 6 10 1 10 7 4 5 8 7 2 2 10 13 4 8 2 11 4 6 14 4 8 2 6 2 3 7 1 12 11 2 9 5 6 10 4 13 4 5 10 4 11 9 3 3 11 9 3 2 3 8 15 6 20 17 19 21 10 15 3 7 11 11 7 17 20 14 9 16 6 17 13 21 21 10 15 22 6 17 21 15 7 17 10 22 22 3 20 8 15 20 16 17 21 10 16 6 22 6 21 14 14 14 16 7 17 3 20 10 7 16 19 14 17 7 21 20 16 7 15 22 10 20 10 18 11 22 18 18 7 19 15 7 22 21 18 7 21 16 3 14 13 7 22 17 13 19 7 8 12 10 17 15 3 21 14 9 7 19 6 15 7 14 14 4 17 10 15 20 19 21 6 18 4 20 16 2 19 8 17 6 13 12 12 6 17 4 20 16 21 12 10 19 16 14 14 15 2 7 21 8 16 21 6 22 16 14 17 22 14 17 20 10 21 7 15 21 18 16 13 20 18 21 12 15 7 4 22 14 13 7 19 14 8 15 4 4 5 3 20 7 16 22 18 6 18 13 20 19 6 16 3 13 3 18 6 22 7 20 18 10 17 11 21 8 13 7 10 17 19 10 14

wherein: (A) each of 1 to 22 is a 4mer selected from the group of 4mers consisting of WWWW, WWWX, WWWY, WWXW, WWXX, WWXY, WWYW, WWYX, WWYY, WXWW, WXWX, WXWY, WXXW, WXXX, WXXY, WXYW, WXYX, WXYY, WYWW, WYWX, WYWY, WYXW, WYXX, WYXY, WYYW, WYYX, WYYY, XWWW, XWWX, XWWY, XWXW, XWXX, XWXY, XWYW, XWYX, XWYY, XXWW, XXWX, XXWY, XXXW, XXXX, XXXY, XXYW, XXYX, XXYY, XYWW, XYWX, XYWY, XYXW, XYXX, XYXY, XYYW, XYYX, XYYY, YWWW, YWWX, YWWY, YWXW, YWXX, YWXY, YWYW, YWYX, YWYY, YXWW, YXWX, YXWY, YXXW, YXXX, YXXY, YXYW, YXYX, YXYY, YYWW, YYWX, YYWY, YYXW, YYXX, YYXY, YYYW, YYYX, and YYYY, and (B) each of 1 to 22 is selected so as to be different from all of the others of 1 to 22; (B) each of W, X and Y is a base in which: (i) (a) W=one of A, T/U, G, and C, X=one of A, T/U, G, and C, Y=one of A, T/U, G, and C, and each of W, X and Y is selected so as to be different from all of the others of W, X and Y,  (b) an unselected said base of (i)(a) can be substituted any number of times for any one of W, X and Y, or (ii) (a) W=G or C, X=A or T/U, Y=A or T/U, and X≠Y, and  (b) a base not selected in (ii)(a) can be inserted into each sequence at one or more locations, the location of each insertion being the same in all the sequences; (D) up to three bases can be inserted at any location of any of the sequences or up to three bases can be deleted from any of the sequences; (E) all of the sequences of a said group of oligonucleotides are read 5′ to 3′ or are read 3′ to 5′; and wherein each oligonucleotide of a said set has a sequence of at least ten contiguous bases of the sequence on which it is based, provided that: (F) (I) the quotient of the sum of G and C divided by the sum of A, T/U, G and C for all combined sequences of the set is between about 0.1 and 0.40 and said quotient for each sequence of the set does not vary from the quotient for the combined sequences by more than 0.2; and (II) for any phantom sequence generated from any pair of first and second sequences of the set L₁ and L₂ in length, respectively, by selection from the first and second sequences of identical bases in identical sequence with each other: (i) any consecutive sequence of bases in the phantom sequence which is identical to a consecutive sequence of bases in each of the first and second sequences from which it is generated is less than ((¾×L)−1) bases in length; (ii) the phantom sequence, if greater than or equal to (⅚×L) in length, contains at least three insertions/deletions or mismatches when compared to the first and second sequences from which it is generated; and (iii) the phantom sequence is not greater than or equal to ( 11/12×L) in length; where L=L₁, or if L₁≠L₂, where L is the greater of L₁ and L₂; and wherein any base present may be substituted by an analogue thereof; v) a second target nucleic acid, distinct from said first target nucleic acid, and having a fourth region, a fifth region and a sixth region, wherein said fourth region is located adjacent to and downstream from said fifth region, and said fifth region is located adjacent to and downstream from said sixth region, said fifth region having a sequence complementary to said 3′ portion of said sequence selected from the group of sequences listed in step (a)(iv), said sixth region having a sequence complementary to said 5′ portion of the sequence selected from the group of sequences in step (a)(iv); vi) a third oligonucleotide having a 5′ portion, a central portion and a 3′ portion, said 5′ portion of said third oligonucleotide having a sequence complementary to said fourth region of said second target nucleic acid, said central portion of said third oligonucleotide having a sequence complementary to said fifth region of said second target nucleic acid, and said 3′ portion of said third oligonucleotide having a sequence that is not base paired to either said second target nucleic acid or said first target nucleic acid and is selected from a set of oligonucleotides based on the group of sequences listed in step (a)(iv) such that said sequence selected is distinct from said sequence selected in step (a)(iv); b) mixing said cleavage means, said first target nucleic acid, said second target nucleic acid, said first, second, and third oligonucleotides to create a reaction mixture under reaction conditions such that at least said 5′ portion of said first oligonucleotide is annealed to said first target nucleic acid and wherein at least said 5′ and central portion of said second oligonucleotide is annealed to said first target nucleic acid so as to create a first cleavage structure and wherein the combined melting temperature of said complementary regions within said 5′ and 3′ portions of said first oligonucleotide when annealed to said first target nucleic acid is greater than the melting temperature of said 5′ and central portion of said first oligonucleotide, wherein cleavage of said first cleavage structure occurs to generate a first non-target cleavage product, and wherein at least said 5′ portion first non-target cleavage product is annealed to said second target nucleic acid and at least said 5′ and central portion of said third oligonucleotide is annealed to said second target nucleic acid so as to create a second cleavage structure and wherein the combined melting temperature of said complementary regions within said 5′ and 3′ portions of said non-target cleavage product when annealed to said second target nucleic acid is greater than the melting temperature of said 5′ and central portion of said third oligonucleotide, wherein cleavage of said second cleavage structure occurs to generate a second non-target cleavage product; and c) detecting said second non-target cleavage product.
 56. The method of claim 55, wherein said first target nucleic acid is genomic DNA and said second target nucleic acid is synthetic DNA.
 57. The method of claim 55, wherein said synthetic DNA has at least one hairpin loop.
 58. The method of claim 57, wherein the method includes a plurality of said first target nucleic acid sequences, a plurality of first oligonucleotide molecules, a plurality of said second oligonucleotide molecules, a plurality of said second target nucleic acid sequences and a plurality of third oligonucleotide molecules.
 59. A method of analyzing a biological sample comprising a plurality of target nucleic acid molecules for the presence of a mutation or polymorphism at a locus of each target nucleic acid molecule, the method comprising: a) providing: i) a cleavage means, ii) a first target nucleic acid, said first target nucleic acid having a first region, a second region and a third region, wherein said first region is located adjacent to and downstream from said second region, and said second region is located adjacent to and downstream from said third region; iii) a first oligonucleotide having a 5′ and a 3′ portion, said 5′ portion of said first oligonucleotide having a sequence complementary to said second region of said target nucleic acid and said 3′ portion of said first oligonucleotide having a sequence complementary to said third region of said target nucleic acid; iv) a second oligonucleotide having a 5′ portion, a central portion and a 3′ portion, said 5′ portion of said second oligonucleotide having a sequence complementary to said first region of said target nucleic acid, said central portion of said second oligonucleotide having a sequence complimentary to said second region of said target nucleic acid, and said 3′ portion of said second oligonucleotide having a sequence that is not base-paired to said target nucleic acid and is selected from a set of oligonucleotides, based on a following group of sequences each having a 3′ and 5′ portion, 1 4 6 6 1 3 2 4 5 5 2 3 1 8 1 2 3 4 1 7 1 9 8 4 1 1 9 2 6 9 1 2 4 3 9 6 9 8 9 8 10 9 9 1 2 3 8 10 8 8 7 4 3 1 1 1 1 1 1 2 2 1 3 3 2 2 3 1 2 2 3 2 4 1 4 4 4 2 1 2 3 3 1 1 1 3 2 2 1 4 3 3 3 3 3 4 4 3 1 1 4 4 3 4 1 1 3 3 3 6 6 6 3 5 6 6 1 1 6 5 7 6 7 7 7 5 8 7 5 5 8 8 2 1 7 7 1 1 2 3 2 3 1 3 2 6 5 6 1 6 4 8 1 1 3 8 5 3 1 1 6 3 5 6 8 8 6 6 8 3 6 5 7 3 1 2 3 1 4 6 1 5 7 5 4 3 2 1 6 7 3 6 2 6 1 3 3 1 2 7 6 8 3 1 3 4 3 1 2 5 3 5 6 1 2 7 3 6 1 7 2 7 4 6 3 5 1 7 5 4 6 3 8 6 6 8 2 3 7 1 7 1 7 8 6 3 7 3 4 1 6 8 4 7 7 1 2 4 3 6 5 2 6 3 1 4 1 4 6 1 3 3 1 4 8 1 8 3 3 5 3 8 1 3 6 6 3 7 7 3 8 6 4 7 3 1 3 7 8 6 10 9 5 5 10 10 7 10 10 10 7 9 9 9 7 7 10 9 9 3 10 3 10 3 9 6 3 4 10 6 10 4 10 3 9 4 3 9 3 10 4 9 9 10 5 9 4 8 3 9 4 9 10 7 3 5 9 4 10 8 4 10 5 4 9 3 5 3 3 9 8 10 6 8 6 9 7 10 4 6 10 9 6 4 4 9 8 10 8 3 7 7 9 10 5 3 8 8 9 3 9 10 8 10 2 9 5 9 9 6 2 2 7 10 9 7 5 3 10 6 10 3 6 8 9 2 10 9 3 2 7 3 8 9 10 3 6 2 3 2 5 10 8 9 8 2 3 10 2 9 6 3 9 8 2 10 3 7 3 9 9 10 9 10 1 1 9 4 10 1 9 1 4 1 7 1 10 9 8 1 9 1 10 1 10 6 9 6 9 1 3 10 3 10 8 8 9 1 3 8 1 9 10 3 9 10 1 3 6 9 1 9 1 10 3 1 1 4 9 6 8 10 3 3 9 6 1 10 5 3 1 6 9 10 6 1 8 10 9 6 5 9 9 4 10 3 2 10 9 1 9 5 10 10 7 2 1 9 10 9 9 1 8 2 1 8 6 8 9 10 1 9 1 3 8 10 9 6 9 10 1 2 1 10 8 9 9 2 1 9 6 7 2 9 4 3 9 3 5 1 5 11 10 14 12 1 7 12 4 13 3 2 5 5 4 4 12 9 2 13 13 11 13 13 10 2 5 4 12 7 11 7 4 11 6 4 12 12 1 9 11 11 12 9 4 14 12 6 12 7 13 2 9 11 9 11 3 4 1 3 10 5 12 11 4 4 4 13 7 12 1 5 9 13 10 11 11 6 10 14 14 10 1 3 2 14 1 10 4 5 10 12 12 7 11 10 9 11 2 12 8 11 2 8 5 2 12 14 1 8 13 3 7 8 9 4 7 5 4 2 13 2 12 7 1 12 11 10 9 7 5 11 8 12 2 2 12 7 5 2 14 3 4 13 1 8 8 1 5 9 14 5 11 10 13 3 14 1 4 13 2 4 4 4 5 11 3 10 10 9 2 3 3 11 11 4 8 14 3 4 5 1 14 8 11 2 14 3 11 6 12 5 13 4 4 1 10 1 6 10 11 6 5 1 5 8 12 5 1 7 4 5 9 6 9 2 13 2 4 4 2 3 11 2 2 5 9 3 8 1 10 12 2 8 12 7 9 11 4 1 12 1 4 14 3 13 11 2 7 10 4 1 3 4 12 11 11 11 3 3 4 2 12 11 1 5 9 4 2 1 6 1 12 2 10 5 10 5 1 12 2 14 2 11 7 9 4 11 7 4 4 5 14 12 12 5 2 1 10 12 5 9 2 11 6 1 12 14 3 6 1 14 5 9 11 10 1 4 2 5 12 14 10 10 4 5 8 4 5 6 10 12 4 6 12 5 4 2 1 13 6 8 9 10 10 14 5 3 6 14 10 11 3 3 2 9 10 12 5 7 13 3 7 10 5 12 6 4 1 2 5 13 6 1 13 4 14 13 2 12 1 14 1 9 4 11 13 2 6 10 1 10 7 4 5 8 7 2 2 10 13 4 8 2 11 4 6 14 4 8 2 6 2 3 7 1 12 11 2 9 5 6 10 4 13 4 5 10 4 11 9 3 3 11 9 3 2 3 8 15 6 20 17 19 21 10 15 3 7 11 11 7 17 20 14 9 16 6 17 13 21 21 10 15 22 6 17 21 15 7 17 10 22 22 3 20 8 15 20 16 17 21 10 16 6 22 6 21 14 14 14 16 7 17 3 20 10 7 16 19 14 17 7 21 20 16 7 15 22 10 20 10 18 11 22 18 18 7 19 15 7 22 21 18 7 21 16 3 14 13 7 22 17 13 19 7 8 12 10 17 15 3 21 14 9 7 19 6 15 7 14 14 4 17 10 15 20 19 21 6 18 4 20 16 2 19 8 17 6 13 12 12 6 17 4 20 16 21 12 10 19 16 14 14 15 2 7 21 8 16 21 6 22 16 14 17 22 14 17 20 10 21 7 15 21 18 16 13 20 18 21 12 15 7 4 22 14 13 7 19 14 8 15 4 4 5 3 20 7 16 22 18 6 18 13 20 19 6 16 3 13 3 18 6 22 7 20 18 10 17 11 21 8 13 7 10 17 19 10 14

wherein: (A) each of 1 to 22 is a 4mer selected from the group of 4mers consisting of WWWW, WWWX, WWWY, WWXW, WWXX, WWXY, WWYW, WWYX, WWYY, WXWW, WXWX, WXWY, WXXW, WXXX, WXXY, WXYW, WXYX, WXYY, WYWW, WYWX, WYWY, WYXW, WYXX, WYXY, WYYW, WYYX, WYYY, XWWW, XWWX, XWWY, XWXW, XWXX, XWXY, XWYW, XWYX, XWYY, XXWW, XXWX, XXWY, XXXW, XXXX, XXXY, XXYW, XXYX, XXYY, XYWW, XYWX, XYWY, XYXW, XYXX, XYXY, XYYW, XYYX, XYYY, YWWW, YWWX, YWWY, YWXW, YWXX, YWXY, YWYW, YWYX, YWYY, YXWW, YXWX, YXWY, YXXW, YXXX, YXXY, YXYW, YXYX, YXYY, YYWW, YYWX, YYWY, YYXW, YYXX, YYXY, YYYW, YYYX, and YYYY, and (B) each of 1 to 22 is selected so as to be different from all of the others of 1 to 22; each of W, X and Y is a base in which: (i) (a) W=one of A, T/U, G, and C, X=one of A, T/U, G, and C, Y=one of A, T/U, G, and C, and each of W, X and Y is selected so as to be different from all of the others of W, X and Y,  (b) an unselected said base of (i)(a) can be substituted any number of times for any one of W, X and Y, or (ii) (a) W=G or C, X=A or T/U, Y=A or T/U, and X═Y, and  (b) a base not selected in (ii)(a) can be inserted into each sequence at one or more locations, the location of each insertion being the same in all the sequences; (D) up to three bases can be inserted at any location of any of the sequences or up to three bases can be deleted from any of the sequences; (E) all of the sequences of a said group of oligonucleotides are read 5′ to 3′ or are read 3′ to 5′; and wherein each oligonucleotide of a said set has a sequence of at least ten contiguous bases of the sequence on which it is based, provided that: (F) (I) the quotient of the sum of G and C divided by the sum of A, T/U, G and C for all combined sequences of the set is between about 0.1 and 0.40 and said quotient for each sequence of the set does not vary from the quotient for the combined sequences by more than 0.2; and (II) for any phantom sequence generated from any pair of first and second sequences of the set L₁ and L₂ in length, respectively, by selection from the first and second sequences of identical bases in identical sequence with each other: (i) any consecutive sequence of bases in the phantom sequence which is identical to a consecutive sequence of bases in each of the first and second sequences from which it is generated is less than ((¾×L)−1) bases in length; (ii) the phantom sequence, if greater than or equal to (⅚×L) in length, contains at least three insertions/deletions or mismatches when compared to the first and second sequences from which it is generated; and (iii) the phantom sequence is not greater than or equal to ( 11/12×L) in length; where L=L₁, or if L₁≠L₂, where L is the greater of L₁ and L₂; and wherein any base present may be substituted by an analogue thereof; v) a second target nucleic acid, distinct from said first target nucleic acid, and having a fourth region, a fifth region and a sixth region, wherein said fourth region is located adjacent to and downstream from said fifth region, and said fifth region is located adjacent to and downstream from said sixth region, said fifth region having a sequence complementary to said 3′ portion of said sequence selected from the group of sequences listed in step (a)(iv), said sixth region having a sequence complementary to said 5′ portion of the sequence selected from the group of sequences in step (a)(iv); vi) a third oligonucleotide having a 5′ portion, a central portion and a 3′ portion, said 5′ portion of said third oligonucleotide having a sequence complementary to said fourth region of said second target nucleic acid, said central portion of said third oligonucleotide having a sequence complementary to said fifth region of said second target nucleic acid, and said 3′ portion of said third oligonucleotide having a sequence that is not base paired to either said second target nucleic acid or said first target nucleic acid and is selected from a set of oligonucleotides based on the group of sequences listed in step (a)(iv) such that said sequence selected is distinct from said sequence selected in step (a)(iv); and b) mixing said cleavage means, said first target nucleic acid, said second target nucleic acid, said first, second, and third oligonucleotides to create a reaction mixture under reaction conditions such that at least said 5′ portion of said first oligonucleotide is annealed to said first target nucleic acid and wherein at least said 5′ and central portion of said second oligonucleotide is annealed to said first target nucleic acid so as to create a first cleavage structure and wherein the combined melting temperature of said complementary regions within said 5′ and 3′ portions of said first oligonucleotide when annealed to said first target nucleic acid is greater than the melting temperature of said 5′ and central portion of said first oligonucleotide, wherein cleavage of said first cleavage structure occurs to generate a first non-target cleavage product, and wherein at least said 5′ portion first non-target cleavage product is annealed to said second target nucleic acid and at least said 5′ and central portion of said third oligonucleotide is annealed to said second target nucleic acid so as to create a second cleavage structure and wherein the combined melting temperature of said complementary regions within said 5′ and 3′ portions of said non-target cleavage product when annealed to said second target nucleic acid is greater than the melting temperature of said 5′ and central portion of said third oligonucleotide, wherein cleavage of said second cleavage structure occurs to generate a second non-target cleavage product; and c) detecting said second non-target cleavage product.
 60. The method of claim 59, wherein said first target nucleic acid is genomic DNA and said second target nucleic acid is synthetic DNA.
 61. The method of claim 60, wherein said synthetic DNA has at least one hairpin loop.
 62. The method of claim 61, wherein the method includes a plurality of said first target nucleic acid sequences, a plurality of first oligonucleotide molecules, a plurality of said second oligonucleotide molecules, a plurality of said second target nucleic acid sequences and a plurality of third oligonucleotide molecules.
 63. A method of detecting the presence of a target nucleic acid molecule by detecting non-target cleavage products, the method comprising: a) providing: i) a cleavage means, ii) a first target nucleic acid, said first target nucleic acid having a first region, a second region and a third region, wherein said first region is located adjacent to and downstream from said second region, and said second region is located adjacent to and downstream from said third region; iii) a first oligonucleotide having a 5′ and a 3′ portion, said 5′ portion of said first oligonucleotide having a sequence complementary to said second region of said target nucleic acid and said 3′ portion of said first oligonucleotide having a sequence complementary to said third region of said target nucleic acid; iv) a second oligonucleotide having a 5′ portion, a central portion and a 3′ portion, said 5′ portion of said second oligonucleotide having a sequence complementary to said first region of said target nucleic acid, said central portion of said second oligonucleotide having a sequence complimentary to said second region of said target nucleic acid, and said 3′ portion of said second oligonucleotide having a sequence that is not base-paired to said target nucleic acid and is selected from a set of oligonucleotides, based on a following group of sequences each having a 3′ and 5′ portion, 1 1 1 2 2 3 2 3 1 1 1 3 1 2 2 3 2 2 2 3 2 3 2 1 3 2 2 1 3 1 3 2 2 1 1 2 2 3 2 1 2 2 2 3 1 2 3 1 1 2 3 2 2 1 1 1 3 2 1 1 3 2 3 2 2 3 1 1 1 2 3 2 2 3 1 2 3 2 2 1 3 1 1 3 2 1 2 1 2 2 3 2 3 1 1 2 2 2 2 3 2 3 2 1 3 1 1 2 1 2 3 2 3 2 2 3 2 2 1 1 1 2 1 1 3 2 3 2 1 1 3 2 3 1 1 1 2 1 1 3 1 1 3 1 1 1 3 1 3 2 1 2 2 2 3 2 2 3 2 3 1 3 2 2 1 1 1 2 3 2 3 2 2 2 1 2 3 2 2 1 2 1 2 3 2 3 1 1 3 2 2 2 1 1 1 3 1 3 1 1 2 1 3 1 1 2 1 2 3 2 3 2 1 1 3 2 2 1 2 3 1 1 1 3 1 3 2 3 1 3 1 2 1 1 2 3 2 2 2 1 1 2 3 1 3 1 1 1 2 1 2 3 2 2 1 3 1 1 2 3 2 3 1 2 2 2 1 3 2 2 3 2 2 3 1 2 3 2 2 2 1 3 2 1 3 2 2 2 3 2 1 1 1 3 1 3 2 1 2 1 1 3 2 2 2 3 1 2 3 1 2 1 1 1 1 3 2 1 1 3 1 1 2 3 1 2 3 2 1 1 2 1 1 3 2 3 3 2 1 3 1 1 1 2 1 3 2 2 2 1 2 2 3 1 2 3 1 2 2 3 2 3 2 1 1 3 2 3 1 1 1 2 1 3 2 3 1 3 2 2 1 2 2 2 1 1 1 2 1 3 1 2 3 1 2 1 2 1 1 3 2 3 1 3 1 1 2 3 1 2 1 1 3 2 2 1 2 1 1 3 2 3 2 2 1 2 3 2 3 1 3 2 2 1 2 1 3 1 2 1 1 1 3 1 3 1 2 3 1 2 2 2 3 2 2 3 1 3 1 3 2 2 3 1 3 1 1 2 3 2 1 2 1 3 2 1 2 2 1 2 1 1 3 2 1 3 2 2 2 3 2 1 1 3 1 1 2 3 1 2 2 3 2 1 2 2 1 2 3 1 1 1 2 2 3 1 3 2 3 1 1 3 1 2 2 3 1 2 3 2 1 2 1 2 3 2 1 1 1 2 2 3 2 2 1 2 3 2 2 3 1 3 3 1 1 2 2 3 2 1 2 1 1 1 3 2 1 2 2 1 3 1 2 3 2 3 2 1 3 1 2 3 1 3 1 2 2 1 1 3 2 3 2 2 1 2 2 2 3 1 3 2 2 1 1 3 2 2 2 3 2 2 2 1 2 3 2 1 2 1 3 1 1 3 3 1 3 2 1 2 2 1 3 2 1 1 1 3 2 3 1 2 1 2 3 1 2 1 3 2 3 1 1 2 3 1 2 2 2 1 3 2 1 1 1 2 3 1 2 2 3 1 3 1 2 2 3 1 1 3 2 2 1 2 1 3 1 1 1 2 3 1 2 2 1 3 1 3 2 3 1 2 1 1 1 2 3 2 2 1 3 2 2 3 1 1 2 2 3 2 2 1 2 1 2 1 3 2 1 1 1 2 3 2 2 2 3 2 3 2 3 2 2 3 2 2 1 1 3 2 3 2 2 1 3 2 2 1 2 2 2 3 2 2 3 2 1 3 3 2 1 3 2 1 1 2 1 2 3 1 1 3 2 3 1 3 1 1 2 1 2 1 2 1 3 2 3 2 1 2 1 3 1 1 2 3 2 1 3 1 2 2 2 1 3 2 2 2 3 2 1 3 1 2 2 1 3 1 2 3 2 3 2 2 2 3 2 1 1 1 2 1 3 2 1 2 1 3 1 3 2 1 3 1 3 1 2 3 1 2 1 2 2 2 1 2 2 3 2 3 1 1 1 3 1 1 1 3 1 3 1 1 3 1 1 1 2 2 2 3 2 3 1 3 1 1 2 2 1 1 3 1 2 2 1 1 3 1 1 2 3 2 1 2 1 2 2 1 3 2 2 1 1 3 1 1 1 3 1 1 3 1 3 2 2 3 2 2 3 2 1 3 2 2 3 1 3 1 1 1 2 1 2 3 2 1 3 2 2 2 2 1 3 1 3 2 2 3 2 2 1 1 1 3 1 3 2 3 2 1 1 1 2 1 3 2 2 1 2 3 1 2 3 2 3 2 1 2 1 1 3 2 1 1 2 1 2 3 2 2 2 3 2 2 1 3 1 1 2 3 1 3 1 1 3 1 2 2 2 1 2 3 1 3 2 1 2 1 3 2 2 2 1 1 1 3 1 1 3 2 1 3 2 1 3 1 3 2 3 1 3 1 2 1 2 1 3 1 2 2 2 1 3 1 1 1 3 2 1 1 2 2 3 2 2 2 1 2 1 3 2 3 1 1 3 2 3 1 1 2 1 3 2 1 1 1 3 2 1 1 3 2 1 3 2 1 1 2 1 3 2 3 2 3 2 2 1 1 1 2 2 2 3 2 3 1 3 2 2 1 2 3 1 1 1 3 1 2 1 1 3 1 3 1 1 1 3 2 1 3 1 3 1 1 2 1 1 1 3 1 2 1 1 3 1 1 1 2 2 2 1 1 3 1 2 2 3 2 2 1 1 3 1 3 2 1 3 1 1 3 3 2 2 2 1 1 1 3 1 2 2 3 2 1 1 3 1 1 2 3 2 3 2 1 2 2 2 3 2 3 1 1 3 1 2 3 1 1 3 2 1 2 2 2 3 2 1 2 2 3 2 3 2 2 2 1 3 1 1 2 2 2 1 3 2 1 2 3 2 3 2 1 3 1 2 1 1 2 3 1 2 2 1 2 1 3 1 1 1 3 2 3 2 2 2 3 3 2 2 1 2 2 2 3 2 1 1 3 2 2 1 1 3 1 2 1 3 2 1 3 1 3 2 2 2 1 2 2 3 1 1 1 3 1 3 2 2 2 3 1 1 2 1 3 2 2 3 2 3 2 2 2 1 2 2 3 2 3 2 1 3 2 2 2 1 1 1 3 1 2 2 3 2 3 1 3 1 1 3 1 2 1 2 3 1 1 1 3 2 2 1 2 2 3 1 3 1 1 2 3 2 1 1 1 3 1 1 2 3 2 2 2 1 2 2 3 1 2 3 2 3 1 1 1 3 2 2 1 2 3 1 2 3 2 2 1 1 2 2 3 3 2 2 2 1 3 2 1 2 2 1 3 2 2 3 2 2 1 1 3 1 2 2 3 3 1 2 2 3 1 2 1 2 2 2 3 1 1 2 3 2 2 2 3 2 2 2 3 2 3 1 1 2 2 3 1 1 1 3 2 3 2 1 1 2 3 2 2 3 2 1 2 3 1 2 2 3 2 1 2 2 3 2 2 3 1 3 1 1 2 1 3 1 1 2 1 1 1 1 2 2 2 3 1 3 1 2 2 2 3 2 3 1 2 1 3 1 3 2 1 3 2 1 1 2 2 1 3 1 2 2 2 3 2 2 2 3 2 2 3 2 2 3 2 3 2 2 2 3 2 1 2 2 3 2 2 1 3 2 3 1 1 2 1 2 1 3 2 1 2 3 2 1 3 2 1 3 2 1 3 1 2 3 2 2 2 1 2 3 1 1 2 2 3 2 2 2 1 1 1 3 1 2 3 1 2 2 3 1 1 3 1 1 1 2 3 2 3 2 3 1 2 1 1 2 3 1 2 3 2 2 1 2 2 2 3 2 3 2 1 1 2 1 3 2 2 3 2 3 1 3 1 1 2 2 2 3 2 1 1 2 2 1 3 1 2 1 3 1 2 3 2 1 1 3 1 3 1 1 1 2 2 3 2 3 1 1 1 1 3 1 2 2 1 1 3 1 3 1 1 3 2 2 1 1 2 1 3 1 3 2 1 3 1 1 3 2 1 1 1 2 2 3 2 3 1 1 2 3 1 1 1 3 1 1 1 1 1 2 3 2 1 1 3 1 1 1 3 1 1 3 1 2 2 3 2 2 3 2 1 2 2 2 3 1 2 2 2 1 2 3 2 3 2 2 1 2 3 2 2 3 1 3 2 3 2 1 2 2 3 1 3 1 1 1 2 2 2 3 1 1 3 1 1 2 3 1 1 3 1 1 2 2 3 2 1 2 3 1 1 1 2 3 1 1 2 2 3 2 1 1 3 2 1 2 2 3 2 1 3 1 1 3 2 1 1 1 3 2 2 1 3 1 1 3 2 2 2 2 1 2 3 2 1 1 2 3 1 2 1 1 3 2 3 2 1 3 2 2 3 1 2 1 2 1 3 2 2 3 1 1 1 2 2 3 2 3 1 2 1 3 2 3 2 1 2 1 1 3 1 1 1 2 2 1 3 1 3 1 3 2 2 3 2 1 1 1 3 3 1 1 2 2 3 2 3 1 1 1 2 3 2 3 1 2 2 3 1 2 1 2 1 1 1 1 2 1 1 3 2 1 3 2 2 2 1 1 2 3 1 3 1 3 1 1 3 3 1 2 2 1 1 1 3 1 1 3 2 1 1 3 2 3 1 1 2 3 2 2 2 2 1 2 3 2 3 2 3 2 2 3 2 2 2 1 3 2 3 2 2 1 2 2 1 3 1 3 2 2 1 2 1 2 3 2 1 3 2 2 1 3 1 3 2 2 1 2 1 3 1 1 1 3 1 1 1 3 1 1 3 2 3 2 2 1 1 3 2 2 1 1 1 2 1 3 2 1 2 2 1 3 2 1 1 3 2 1 2 3 2 3 1 2 2 3 2 2 2 3 2 3 2 3 1 2 2 3 1 1 2 1 2 2 3 2 3 1 1 1 2 1 2 3 2 3 1 1 1 3 1 3 2 2 1 1 3 2 3 1 2 2 1 1 1 3 1 2 2 3 1 1 2 3 1 2 2 3 1 3 1 2 1 2 3 2 1 1 1 1 1 3 1 2 3 1 2 1 3 2 2 1 1 3 2 3 2 1 1 3 2 2 1 2 1 3 2 2 3 2 2 1 2 2 3 1 3 1 1 2 2 2 1 3 1 1 3 2 2 2 1 2 1 3 2 3 1 1 2 2 1 2 3 1 3 2 3 1 1 1 3 3 1 2 1 3 1 2 2 2 1 3 1 1 2 3 1 1 2 2 1 1 3 2 3 2 2 2 3 1 1 3 1 1 3 1 3 1 2 2 2 3 1 1 1 2 2 3 1 1 2 3 1 1 2 1 1 3 1 3 2 2 3 1 2 1 1 1 2 3 2 3 1 2 3 2 2 2 1 2 3 2 1 3 2 3 2 1 3 1 2 2 3 1 1 2 2 2 2 2 1 1 3 2 3 1 3 2 2 1 2 1 3 1 1 3 2 1 3 2 1 3 1 2 2 2 1 2 3 2 3 2 2 2 3 1 1 3 2 2 1 1 3 1 2 2 1 3 2 2 1 3 1 3 1 1 1 3 2 3 1 2 1 1 1 3 2 2 1 3 2 1 1 2 3 1 2 1 1 2 3 1 1 3 2 3 2 1 2 1 2 1 3 1 1 2 3 1 1 3 2 3 2 2 1 3 2 1 2 1 3 1 2 1 3 2 1 2 1 1 1 2 2 3 1 3 2 2 2 3 2 2 2 3 1 2 2 3 2 1 3 2 1 1 2 3 1 1 3 1 1 2 1 1 3 2 1 2 3 1 3 2 3 2 2 1 1 1 2 3 2 1 1 2 1 3 2 3 2 2 3 2 2 1 3 2 2 1 3 1 3 1 3 2 2 1 3 2 3 1 1 1 2 3 2 2 3 2 2 1 1 1 2 3 1 1 1 2 1 3 1 1 1 2 3 2 1 2 2 3 2 2 2 3 2 3 1 1 3 2 2 1 2 1 1 3 2 2 2 3 2 3 1 3 1 1 2 2 1 1 3 3 1 3 2 2 2 1 2 1 3 2 2 1 3 1 1 2 1 2 3 2 2 3 2 1 3 1 3 2 2 1 2 2 1 3 1 1 3 1 1 3 1 2 2 2 1 1 3 3 1 3 2 2 1 1 2 3 1 1 1 2 1 1 3 2 1 2 2 2 3 2 3 1 2 3 1 2 3 1 1 2 1 3 2 2 3 1 1 3 2 1 2 1 2 1 3 1 2 1 3 1 2 1 2 3 1 3 1 2 3 1 1 1 3 2 2 1 3 2 1 2 1 2 3 2 1 1 1 3 1 1 1 3 2 3 1 1 1 3 1 1 3 1 1 2 3 1 1 2 3 2 1 3 1 1 1 2 3 1 1 2 3 2 2 3 1 1 1 1 1 2 2 3 1 1 2 1 3 2 3 2 3 2 3 1 3 2 2 2 1 1 2 1 3 1 2 1 2 2 3 2 2 2 3 1 2 2 1 1 2 3 1 1 3 1 3 1 1 1 3 2 2 3 2 1 1 1 3 2 2 3 1 1 3 1 2 1 1 1 3 3 2 2 1 1 3 1 3 1 2 2 1 2 3 1 3 1 2 3 2 1 2 2 1 1 3 1 1 3 1 2 1 2 1 1 3 1 1 3 1 2 2 3 1 1 2 2 3 3 2 1 3 1 1 1 2 2 2 3 1 1 2 2 3 1 2 3 2 3 1 1 1 1 1 3 1 3 2 1 3 1 2 2 3 1 2 1 1 3 2 1 2 1 2 3 1 2 3 1 2 1 2 1 3 2 1 3 2 3 1 1 3 1 1 1 2 1 1 3 2 1 3 1 2 1 1 2 3 1 2 3 1 3 1 1 1 2 3 1 1 3 1 2 1 1 2 3 2 3 1 1 1 3 2 1 2 2 2 3 2 3 1 2 1 2 1 3 2 1 1 2 1 1 3 1 3 1 1 2 2 3 1 2 1 2 3 1 1 3 1 2 3 2 1 1 3 2 3 2 1 2 2 2 1 3 2 1 3 1 1 2 3 1 1 3 2 2 1 2 3 2 2 1 3 1 2 2 2 3 2 2 3 1 3 1 2 2 3 1 2 1 3 2 2 2 3 2 1 2 3 1 1 3 1 3 1 2 1 3 2 1 2 2 2 3 1 3 1 1 1 2 3 2 2 1 2 3 2 1 2 2 2 1 3 2 1 3 2 2 1 2 3 2 3 1 3 1 1 2 3 2 3 2 2 2 3 1 2 2 2 1 1 3 2 1 2 3 2 2 2 3 2 2 2 1 2 1 3 1 1 2 3 2 1 2 3 3 1 3 2 1 2 1 2 1 3 1 1 3 1 1 1 3 1 1 1 2 2 2 3 1 2 3 1 3 2 3 1 1 3 2 1 1 1 2 3 2 1 3 2 2 1 2 2 2 2 1 1 3 1 1 3 2 3 1 3 2 2 1 2 2 3 2 3 1 2 1 2 1 2 3 1 1 1 2 3 1 3 1 1 2 1 2 2 3 2 2 3 2 2 2 3 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2 3 2 2 3 1 1 2 2 2 1 3 1 2 3 2 1 3 1 2 1 2 3 1 1 2 3 2 3 1 2 1 3 1 1 3 2 3 2 1 2 2 1 1 3 2 1 1 3 2 2 1 2 1 2 3 1 1 2 2 1 2 3 1 3 1 1 3 1 1 2 1 3 1 3 2 2 2 2 3 2 2 1 2 3 1 1 3 2 3 1 2 2 2 3 2 2 2 3 2 3 2 1 1 1 3 1 2 2 3 2 3 2 2 1 2 1 2 3 1 1 1 2 3 2 2 3 2 3 1 2 1 3 2 1 3 2 2 1 3 1 2 1 2 2 2 3 2 3 1 1 2 2 1 1 3 1 2 1 1 1 3 1 1 3 1 3 1 1 3 2 1 3 1 2 2 3 2 1 3 1 1 2 3 1 1 2 2 2 3 2 1 3 2 1 2 1 1 1 2 1 1 3 1 3 1 3 1 3 1 1 2 3 1 2 2 2 1 3 2 1 1 2 2 1 2 3 2 3 1 1 2 1 3 1 2 2 3 2 2 3 1 1 3 2 2 1 1 3 1 2 2 2 1 2 3 2 3 1 2 1 3 2 1 3 1 3 2 2 2 1 1 1 3 1 2 1 3 2 3 2 2 2 3 2 2 3 2 3 2 2 1 2 1 2 2 3 1 2 2 2 1 2 3 1 1 3 1 3 2 1 2 1 3 2 3 1 1 1 2 2 2 3 1 2 3 1 3 2 1 3 2 2 2 1 1 3 1 3 1 1 2 1 1 1 3 2 2 3 2 2 2 3 1 2 3 2 2 2 3 1 1 2 3 3 1 2 2 3 2 3 1 2 3 1 1 2 1 1 2 3 2 2 1 2 2 3 1 3 1 2 3 1 1 3 1 1 1 2 1 2 3 1 2 1 2 3 1 1 2 1 3 2 2 1 1 1 3 2 2 1 2 2 3 1 1 3 2 3 1 1 3 2 2 3 1 2 2 3 2 1 1 3 1 1 1 2 1 3 1 3 1 2 2 2 3 2 3 2 2 3 1 3 1 2 2 3 1 3 2 2 2 1 1 3 2 1 2 2 1 3 1 2 2 1 3 2 3 1 2 1 1 2 1 3 1 1 2 3 1 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2 1 2 3 1 3 2 2 3 1 2 2 2 3 1 3 2 1 1 3 2 2 2 1 2 1 3 1 2 3 1 1 1 1 3 1 2 1 1 1 3 2 3 1 3 2 2 3 1 2 2 2 1 3 1 2 3 1 2 1 2 2 3 2 1 1 3 1 2 1 2 3 2 2 3 2 1 1 1 3 3 2 1 1 3 1 3 2 3 2 1 2 2 3 2 1 1 3 2 2 1 1 2 2 3 2 3 2 3 1 2 2 1 3 2 1 1 2 3 1 1 3 2 1 2 2 2 1 3 2 1 1 3 1 1 1 3 1 2 2 1 1 3 2 3 2 2 1 3 2 1 1 1 3 2 1 3 1 1 1 3 2 2 3 1 1 1 2 2 3 1 2 2 1 2 3 2 1 1 3 1 3 1 1 3 2 2 3 1 3 2 1 1 2 3 2 1 2 2 2 3 2 2 1 1 3 1 1 1 2 1 3 2 1 3 1 2 1 1 3 2 3 1 1 2 1 1 3 2 1 1 1 2 2 3 1 1 1 3 2 3 2 1 2 1 3 2 3 1 1 3 1 2 3 2 1 2 3 2 2 2 1 2 2 3 2 2 3 2 3 2 1 1 2 2 2 1 3 1 1 2 1 2 1 3 2 3 1 1 3 1 3 1 2 1 3 3 2 2 1 2 3 1 1 1 3 1 3 2 1 2 3 2 3 2 2 1 1 1 2 2 1 2 2 1 2 3 2 3 1 1 3 1 1 3 1 1 2 3 1 2 2 1 3 2 1 1 2 1 1 3 2 2 3 1 1 3 1 3 1 1 2 2 3 2 2 3 2 2 3 1 2 3 2 2 2 3 1 2 3 2 1 1 2 2 3 2 2 1 1 1 3 3 2 3 1 1 1 3 1 2 2 2 3 1 3 2 2 2 3 2 1 2 1 1 2 1 3 1 3 1 1 2 1 2 1 3 1 2 2 3 1 3 1 2 2 2 3 2 2 2 2 2 3 1 3 1 2 3 2 3 1 2 3 1 2 1 1 1 3 2 2 1 1 3 2 2 3 2 1 1 1 2 2 3 2 1 3 2 1 1 1 3 1 1 3 2 1 3 2 3 2 2 1 2 3 1 2 3 2 2 3 2 2 2 3 2 1 2 2 1 2 1 2 2 1 2 2 3 2 3 2 1 3 1 2 3 2 1 2 2 1 1 3 1 3 3 2 2 1 3 1 1 1 3 1 2 2 2 1 3 1 1 3 2 2 1 3 2 2 2 2 3 2 3 2 1 2 2 1 1 3 1 3 1 3 2 3 1 1 1 2 1 2 3 2 2 2 1 1 3 1 2 1 3 1 1 1 3 1 3 2 3 1 2 2 2 1 1 1 2 3 1 3 1 1 1 2 1 3 1 2 1 3 2 2 1 2 2 3 2 3 2 3 1 1 2 2 3 1 1 2 1 1 3 1 1 2 2 2 3 2 2 3 2 3 1 1 2 1 1 3 1 2 2 3 1 1 2 2 1 3 2 3 1 3 2 1 1 3 1 1 2 3 2 2 2 3 1 3 1 3 1 2 2 2 1 3 2 1 1 1 3 1 1 3 2 2 2 1 3 1 1 2 1 3 1 1 1 2 3 2 3 2 2 2 3 1 2 1 2 1 1 3 2 1 1 3 2 3 2 2 1 1 3 1 2 2 2 3 1 3 3 2 1 3 2 3 1 1 2 1 1 3 2 2 1 3 2 3 2 2 1 1 2 1 1 1 3 2 3 2 3 2 2 1 1 1 3 2 1 1 1 2 3 2 1 3 1 2 1 3 1 3 1 2 3 2 2 2 1 2 3 2 2 3 2 3 1 1 2 2 1 1 1 3 2 2 3 1 1 2 1 2 2 3 1 2 3 1 2 1 1 3 1 1 3 1 2 3 1 1 2 3 2 3 1 3 1 2 3 2 2 2 1 3 1 1 2 1 1 2 1 1 2 1 1 2 3 1 2 3 2 1 1 3 2 2 2 3 1 3 2 2 2 3 1 1 1 3 1 3 2 3 1 1 2 1 3 1 1 1 2 1 1 3 1 3 1 1 1 1 2 1 1 1 3 2 2 1 2 2 3 1 3 1 3 1 3 2 2 2 1 3 1 3 2 1 3 2 3 2 2 3 2 1 3 2 2 2 1 3 2 1 2 1 2 1 3 2 1 1 3 1 1 2 3 2 1 2 2 1 3 1 2 1 2 2 2 3 2 3 3 1 2 1 1 1 2 3 2 2 2 3 1 2 1 1 1 3 2 1 3 2 2 3 1 2 1 3 2 1 2 3 2 1 2 3 2 3 2 3 1 1 3 1 2 2 2 1 1 2 3 1 1 2 3 2 1 3 1 3 2 3 1 2 2 1 3 2 2 2 1 1 3 2 1 3 2 1 2 2 2 1 3 2 3 1 2 3 2 1 1 3 1 1 2 1 1 3 1 1 2 2 3 2 1 2 2 3 1 1 3 1 1 3 1 1 2 1 2 3 2 2 2 1 2 1 3 1 1 2 2 3 1 3 1 3 1 1 3 2 2 1 1 3 1 1 1 3 2 1 3 2 1 3 1 3 1 2 2 2 3 1 3 1 1 2 2 1 2 2 2 1 1 1 3 1 1 1 3 2 1 2 2 3 2 1 1 3 1 3 2 3 1 1 1 2 2 3 1 3 1 1 1 3 2 3 1 1 2 3 1 1 3 2 2 2 1 1 3 1 1 1 2 1 1 3 2 1 2 3 1 2 1 3 2 1 3 2 1 3 1 2 2 2 3 1 1 2 2 3 2 1 2 2 3 2 1 3 2 2 2 3 2 3 1 1 3 1 3 1 1 2 1 1 2 3 2 1 3 1 3 1 2 1 2 1 1 3 2 3 2 3 2 1 1 2 1 3 2 2 3 2 2 1 1 2 3 1 3 2 1 1 2 1 2 1 3 2 2 3 2 1 3 2 2 2 1 3 1 2 3 1 1 2 3 2 1 2 2 3 2 3 2 2 1 3 1 1 2 3 1 2 3 2 2 1 1 2 1 3 3 2 2 2 3 2 1 2 1 3 2 1 2 2 2 3 1 2 2 3 1 2 3 2 1 3 1 3 2 1 1 1 3 2 1 2 3 1 3 2 2 1 2 3 1 1 2 1 3 1 1 1 3 2 2 2 1 1 3 2 3 1 2 3 2 1 2 1 2 2 3 2 2 2 1 1 1 2 3 1 2 1 1 1 3 1 3 2 1 3 2 3 1 1 3 2 2 2 1 1 1 2 3 2 3 2 3 1 3 1 1 3 1 2 3 1 1 2 1 1 1 2 2 2 3 2 1 2 1 1 1 3 2 3 1 1 3 1 1 3 1 3 1 1 2 3 1 2 2 1 3 2 1 2 2 2 3 2 3 1 1 3 1 3 1 2 2 2 2 2 2 3 1 1 2 3 1 1 1 2 2 3 1 2 3 1 2 1 3 1 2 3 1 3 1 3 2 1 1 3 1 2 2 1 1 3 1 1 2 1 1 3 1 1 1 3 1 2 2 3 1 1 2 2 3 1 3 1 1 3 2 3 1 1 3 2 1 1 1 2 2 2 2 1 3 1 3 1 1 3 2 1 2 2 3 2 2 2 3 1 1 1 3 1 2 1 1 1 3 2 3 1 1 1 3 1 2 2 2 3 1 1 1 2 3 1 2 3 3 1 1 1 3 2 2 1 3 1 3 1 1 1 2 3 2 1 3 1 1 1 2 2 3 2 3 1 1 2 1 1 2 3 1 1 3 1 1 3 2 2 1 2 3 2 2 1 2 2 3 2 3 1 1 2 1 1 1 3 2 1 3 1 2 3 2 3 2 2 1 2 2 2 1 2 1 2 3 1 2 1 2 3 1 3 2 2 2 3 2 3 2 2 3 1 2 2 3 1 2 2 2 3 2 3 2 3 1 3 2 1 2 2 1 3 2 2 1 2 1 1 1 3 2 3 1 2 2 1 1 3 2 2 1 3 2 2 2 3 1 3 1 2 2 2 3 2 1 2 2 2 3 2 1 2 1 1 2 3 2 2 3 1 1 3 1 3 3 2 2 2 3 1 1 1 2 2 1 3 2 3 2 3 1 3 1 1 1 2 1 2 1 1 2 3 2 2 3 1 3 1 2 2 3 1 2 1 1 2 3 2 2 3 1 1 2 1 3 2 1 3 2 1 3 2 1 2 2 3 2 2 3 2 1 1 2 1 1 3 3 2 2 3 2 1 1 2 2 2 3 1 3 2 3 2 2 1 3 2 2 1 2 2 2 3 1 1 2 1 2 3 1 2 1 3 2 2 1 3 2 1 1 2 2 3 2 3 2 3 1 2 1 3 2 1 2 3 2 2 2 3 2 3 1 2 2 1 1 1 3 1 3 1 2 3 2 1 2 1 1 1 3 1 3 2 1 2 3 2 2 1 2 1 1 3 1 3 2 3 1 3 1 2 2 2 1 3 1 1 3 1 2 3 2 2 1 2 2 1 1 2 3 1 3 1 1 2 2 2 3 2 2 1 1 1 3 1 3 1 1 1 3 2 1 1 1 3 1 1 2 2 1 3 2 1 2 3 1 2 1 3 1 2 3 1 3 1 2 1 3 1 3 2 2 3 2 1 2 1 3 2 2 2 1 2 1 3 2 2 3 1 3 2 1 3 1 1 2 3 1 2 2 3 2 2 2 1 3 1 1 3 1 2 2 2 3 2 2 2 1 2 3 2 2 2 3 1 3 1 1 3 1 3 2 2 1 2 2 2 2 1 3 1 1 3 2 2 2 3 1 1 1 3 2 2 1 2 2 3 1 2 2 3 3 1 2 3 1 1 3 1 3 2 1 2 2 2 3 2 2 1 2 1 2 3 2 1 3 1 2 3 1 1 2 1 2 1 3 2 1 1 3 2 1 2 2 3 1 3 2 1 2 1 3 2 3 1 2 3 1 1 1 2 2 2 3 1 3 1 2 1 3 1 2 1 3 1 1 1 3 1 1 1 2 2 3 1 1 3 1 3 2 2 2 3 1 2 1 2 1 2 2 2 3 1 3 2 1 2 2 2 3 2 3 2 1 2 2 3 1 1 2 3 1 2 3 1 3 2 2 3 1 1 1 2 2 2 3 1 1 3 2 1 2 2 3 2 2 2 1 1 2 1 3 2 3 1 3 1 3 1 3 2 1 2 1 2 3 2 1 1 1 2 2 1 1 3 1 3 1 3 2 3 1 3 2 1 1 1 2 3 2 1 1 1 1 1 3 1 1 2 1 3 1 2 3 1 3 1 2 2 1 3 1 1 1 2 1 3 1 3 2 2 2 1 1 1 3 1 3 2 2 1 3 1 1 2 2 3 1 1 1 3 3 2 1 1 3 1 2 2 2 3 2 2 3 1 1 2 1 1 1 3 1 1 3 1 1 3 1 3 1 1 1 3 1 1 3 2 2 1 1 1 3 2 3 1 2 1 2 2 2 1 1 2 1 3 1 3 1 1 3 1 3 1 2 3 2 1 2 3 1 1 2 1 2 2 1 2 2 1 3 2 3 1 2 1 1 3 2 3 1 1 3 2 2 2 1 3 1 2 1 1 2 3 2 1 1 1 3 1 2 3 1 3 2 2 2 1 2 3 1 3 2 2 3 1 2 2 2 3 1 3 1 3 2 2 3 1 2 1 1 3 1 2 2 2 1 2 3 1 2 2 1 2 2 3 2 3 2 3 2 1 3 1 1 2 2 1 3 1 2 1 2 1 1 1 3 1 2 1 2 1 3 2 1 3 1 2 3 1 2 3 2 3 2 2 2 1 3 2 2 3 1 3 1 2 3 1 1 3 2 2 1 2 2 1 3 1 1 2 2 3 1 1 2 2 3 1 2 1 2 1 3 2 3 2 1 1 1 3 2 3 3 1 1 3 1 1 1 3 1 2 2 1 2 2 3 2 1 2 2 3 1 3 2 2 1 2 2 3 1 3 2 3 2 1 3 2 3 1 2 2 2 1 3 1 1 1 2 1 1 1 2 1 1 1 3 2 3 2 2 2 1 1 3 1 3 2 1 3 1 3 2 1 3 2 1 3 1 3 1 2 1 1 2 2 3 1 2 3 2 3 2 1 1 2 2 2

Wherein each of 1 to 3 is a nucleotide base selected to be different from the others of 1 to 3 with the proviso that up to three nucleotide bases of each sequence can be substituted with any nucleotide base provided that: for any pair of sequences of the set: M1≦16, M2≦13, M3≦20, M4≦16, and M5≦19, where: M1 is the maximum number of matches for any alignment in which there are no internal indels; M2 is the maximum length of a block of matches for any alignment; M3 is the maximum number of matches for any alignment having a maximum score; M4 is the maximum sum of the lengths of the longest two blocks of matches for any alignment of maximum score; and M5 is the maximum sum of the lengths of all the blocks of matches having a length of at least 3, for any alignment of maximum score; wherein: the score of an alignment is determined according to the equation (A×m)−(B×mm)−(C×(og+eg))−(D×eg)), wherein: for each of (i) to (iv):  (i) m=6, mm=6, og=0 and eg=6,  (ii) m=6, mm=6, og=5 and eg=1,  (iii) m=6, mm=2, og=5 and eg=1, and  (iv) m=6, mm=6, og=6 and eg=0, A is the total number of matched pairs of bases in the alignment; B is the total number of internal mismatched pairs in the alignment; C is the total number of internal gaps in the alignment; and D is the total number of internal indels in the alignment minus the total number of internal gaps in the alignment; and wherein the maximum score is determined separately for each of (i), (ii), (iii) and (iv). v) a second target nucleic acid, distinct from said first target nucleic acid, and having a fourth region, a fifth region and a sixth region, wherein said fourth region is located adjacent to and downstream from said fifth region, and said fifth region is located adjacent to and downstream from said sixth region, said fifth region having a sequence complementary to said 3′ portion of said sequence selected from the group of sequences listed in step (a)(iv), said sixth region having a sequence complementary to said 5′ portion of the sequence selected from the group of sequences in step (a)(iv); vi) a third oligonucleotide having a 5′ portion, a central portion and a 3′ portion, said 5′ portion of said third oligonucleotide having a sequence complementary to said fourth region of said second target nucleic acid, said central portion of said third oligonucleotide having a sequence complementary to said fifth region of said second target nucleic acid, and said 3′ portion of said third oligonucleotide having a sequence that is not base paired to either said second target nucleic acid or said first target nucleic acid and is selected from a set of oligonucleotides based on the group of sequences listed in step (a)(iv) such that said sequence selected is distinct from said sequence selected in step (a)(iv); b) mixing said cleavage means, said first target nucleic acid, said second target nucleic acid, said first, second, and third oligonucleotides to create a reaction mixture under reaction conditions such that at least said 5′ portion of said first oligonucleotide is annealed to said first target nucleic acid and wherein at least said 5′ and central portion of said second oligonucleotide is annealed to said first target nucleic acid so as to create a first cleavage structure and wherein the combined melting temperature of said complementary regions within said 5′ and 3′ portions of said first oligonucleotide when annealed to said first target nucleic acid is greater than the melting temperature of said 5′ and central portion of said first oligonucleotide, wherein cleavage of said first cleavage structure occurs to generate a first non-target cleavage product, and wherein at least said 5′ portion first non-target cleavage product is annealed to said second target nucleic acid and at least said 5′ and central portion of said third oligonucleotide is annealed to said second target nucleic acid so as to create a second cleavage structure and wherein the combined melting temperature of said complementary regions within said 5′ and 3′ portions of said non-target cleavage product when annealed to said second target nucleic acid is greater than the melting temperature of said 5′ and central portion of said third oligonucleotide, wherein cleavage of said second cleavage structure occurs to generate a second non-target cleavage product; and c) detecting said second non-target cleavage product.
 64. The method of claim 63, wherein said first target nucleic acid is genomic DNA and said second target nucleic acid is synthetic DNA.
 65. The method of claim 64, wherein said synthetic DNA has at least one hairpin loop.
 66. The method of claim 65, wherein the method includes a plurality of said first target nucleic acid sequences, a plurality of first oligonucleotide molecules, a plurality of said second oligonucleotide molecules, a plurality of said second target nucleic acid sequences and a plurality of third oligonucleotide molecules.
 67. A method of analyzing a biological sample comprising a plurality of target nucleic acid molecules for the presence of a mutation or polymorphism at a locus of each target nucleic acid molecule, the method comprising: a) providing: i) a cleavage means, ii) a first target nucleic acid, said first target nucleic acid having a first region, a second region and a third region, wherein said first region is located adjacent to and downstream from said second region, and said second region is located adjacent to and downstream from said third region; iii) a first oligonucleotide having a 5′ and a 3′ portion, said 5′ portion of said first oligonucleotide having a sequence complementary to said second region of said target nucleic acid and said 3′ portion of said first oligonucleotide having a sequence complementary to said third region of said target nucleic acid; iv) a second oligonucleotide having a 5′ portion, a central portion and a 3′ portion, said 5′ portion of said second oligonucleotide having a sequence complementary to said first region of said target nucleic acid, said central portion of said second oligonucleotide having a sequence complimentary to said second region of said target nucleic acid, and said 3′ portion of said 20 second oligonucleotide having a sequence that is not base-paired to said target nucleic acid and is selected from a set of oligonucleotides, based on a following group of sequences each having a 3′ and 5′ portion, 1 1 1 2 2 3 2 3 1 1 1 3 1 2 2 3 2 2 2 3 2 3 2 1 3 2 2 1 3 1 3 2 2 1 1 2 2 3 2 1 2 2 2 3 1 2 3 1 1 2 3 2 2 1 1 1 3 2 1 1 3 2 3 2 2 3 1 1 1 2 3 2 2 3 1 2 3 2 2 1 3 1 1 3 2 1 2 1 2 2 3 2 3 1 1 2 2 2 2 3 2 3 2 1 3 1 1 2 1 2 3 2 3 2 2 3 2 2 1 1 1 2 1 1 3 2 3 2 1 1 3 2 3 1 1 1 2 1 1 3 1 1 3 1 1 1 3 1 3 2 1 2 2 2 3 2 2 3 2 3 1 3 2 2 1 1 1 2 3 2 3 2 2 2 1 2 3 2 2 1 2 1 2 3 2 3 1 1 3 2 2 2 1 1 1 3 1 3 1 1 2 1 3 1 1 2 1 2 3 2 3 2 1 1 3 2 2 1 2 3 1 1 1 3 1 3 2 3 1 3 1 2 1 1 2 3 2 2 2 1 1 2 3 1 3 1 1 1 2 1 2 3 2 2 1 3 1 1 2 3 2 3 1 2 2 2 1 3 2 2 3 2 2 3 1 2 3 2 2 2 1 3 2 1 3 2 2 2 3 2 1 1 1 3 1 3 2 1 2 1 1 3 2 2 2 3 1 2 3 1 2 1 1 1 1 3 2 1 1 3 1 1 2 3 1 2 3 2 1 1 2 1 1 3 2 3 3 2 1 3 1 1 1 2 1 3 2 2 2 1 2 2 3 1 2 3 1 2 2 3 2 3 2 1 1 3 2 3 1 1 1 2 1 3 2 3 1 3 2 2 1 2 2 2 1 1 1 2 1 3 1 2 3 1 2 1 2 1 1 3 2 3 1 3 1 1 2 3 1 2 1 1 3 2 2 1 2 1 1 3 2 3 2 2 1 2 3 2 3 1 3 2 2 1 2 1 3 1 2 1 1 1 3 1 3 1 2 3 1 2 2 2 3 2 2 3 1 3 1 3 2 2 3 1 3 1 1 2 3 2 1 2 1 3 2 1 2 2 1 2 1 1 3 2 1 3 2 2 2 3 2 1 1 3 1 1 2 3 1 2 2 3 2 1 2 2 1 2 3 1 1 1 2 2 3 1 3 2 3 1 1 3 1 2 2 3 1 2 3 2 1 2 1 2 3 2 1 1 1 2 2 3 2 2 1 2 3 2 2 3 1 3 3 1 1 2 2 3 2 1 2 1 1 1 3 2 1 2 2 1 3 1 2 3 2 3 2 1 3 1 2 3 1 3 1 2 2 1 1 3 2 3 2 2 1 2 2 2 3 1 3 2 2 1 1 3 2 2 2 3 2 2 2 1 2 3 2 1 2 1 3 1 1 3 3 1 3 2 1 2 2 1 3 2 1 1 1 3 2 3 1 2 1 2 3 1 2 1 3 2 3 1 1 2 3 1 2 2 2 1 3 2 1 1 1 2 3 1 2 2 3 1 3 1 2 2 3 1 1 3 2 2 1 2 1 3 1 1 1 2 3 1 2 2 1 3 1 3 2 3 1 2 1 1 1 2 3 2 2 1 3 2 2 3 1 1 2 2 3 2 2 1 2 1 2 1 3 2 1 1 1 2 3 2 2 2 3 2 3 2 3 2 2 3 2 2 1 1 3 2 3 2 2 1 3 2 2 1 2 2 2 3 2 2 3 2 1 3 3 2 1 3 2 1 1 2 1 2 3 1 1 3 2 3 1 3 1 1 2 1 2 1 2 1 3 2 3 2 1 2 1 3 1 1 2 3 2 1 3 1 2 2 2 1 3 2 2 2 3 2 1 3 1 2 2 1 3 1 2 3 2 3 2 2 2 3 2 1 1 1 2 1 3 2 1 2 1 3 1 3 2 1 3 1 3 1 2 3 1 2 1 2 2 2 1 2 2 3 2 3 1 1 1 3 1 1 1 3 1 3 1 1 3 1 1 1 2 2 2 3 2 3 1 3 1 1 2 2 1 1 3 1 2 2 1 1 3 1 1 2 3 2 1 2 1 2 2 1 3 2 2 1 1 3 1 1 1 3 1 1 3 1 3 2 2 3 2 2 3 2 1 3 2 2 3 1 3 1 1 1 2 1 2 3 2 1 3 2 2 2 2 1 3 1 3 2 2 3 2 2 1 1 1 3 1 3 2 3 2 1 1 1 2 1 3 2 2 1 2 3 1 2 3 2 3 2 1 2 1 1 3 2 1 1 2 1 2 3 2 2 2 3 2 2 1 3 1 1 2 3 1 3 1 1 3 1 2 2 2 1 2 3 1 3 2 1 2 1 3 2 2 2 1 1 1 3 1 1 3 2 1 3 2 1 3 1 3 2 3 1 3 1 2 1 2 1 3 1 2 2 2 1 3 1 1 1 3 2 1 1 2 2 3 2 2 2 1 2 1 3 2 3 1 1 3 2 3 1 1 2 1 3 2 1 1 1 3 2 1 1 3 2 1 3 2 1 1 2 1 3 2 3 2 3 2 2 1 1 1 2 2 2 3 2 3 1 3 2 2 1 2 3 1 1 1 3 1 2 1 1 3 1 3 1 1 1 3 2 1 3 1 3 1 1 2 1 1 1 3 1 2 1 1 3 1 1 1 2 2 2 1 1 3 1 2 2 3 2 2 1 1 3 1 3 2 1 3 1 1 3 3 2 2 2 1 1 1 3 1 2 2 3 2 1 1 3 1 1 2 3 2 3 2 1 2 2 2 3 2 3 1 1 3 1 2 3 1 1 3 2 1 2 2 2 3 2 1 2 2 3 2 3 2 2 2 1 3 1 1 2 2 2 1 3 2 1 2 3 2 3 2 1 3 1 2 1 1 2 3 1 2 2 1 2 1 3 1 1 1 3 2 3 2 2 2 3 3 2 2 1 2 2 2 3 2 1 1 3 2 2 1 1 3 1 2 1 3 2 1 3 1 3 2 2 2 1 2 2 3 1 1 1 3 1 3 2 2 2 3 1 1 2 1 3 2 2 3 2 3 2 2 2 1 2 2 3 2 3 2 1 3 2 2 2 1 1 1 3 1 2 2 3 2 3 1 3 1 1 3 1 2 1 2 3 1 1 1 3 2 2 1 2 2 3 1 3 1 1 2 3 2 1 1 1 3 1 1 2 3 2 2 2 1 2 2 3 1 2 3 2 3 1 1 1 3 2 2 1 2 3 1 2 3 2 2 1 1 2 2 3 3 2 2 2 1 3 2 1 2 2 1 3 2 2 3 2 2 1 1 3 1 2 2 3 3 1 2 2 3 1 2 1 2 2 2 3 1 1 2 3 2 2 2 3 2 2 2 3 2 3 1 1 2 2 3 1 1 1 3 2 3 2 1 1 2 3 2 2 3 2 1 2 3 1 2 2 3 2 1 2 2 3 2 2 3 1 3 1 1 2 1 3 1 1 2 1 1 1 1 2 2 2 3 1 3 1 2 2 2 3 2 3 1 2 1 3 1 3 2 1 3 2 1 1 2 2 1 3 1 2 2 2 3 2 2 2 3 2 2 3 2 2 3 2 3 2 2 2 3 2 1 2 2 3 2 2 1 3 2 3 1 1 2 1 2 1 3 2 1 2 3 2 1 3 2 1 3 2 1 3 1 2 3 2 2 2 1 2 3 1 1 2 2 3 2 2 2 1 1 1 3 1 2 3 1 2 2 3 1 1 3 1 1 1 2 3 2 3 2 3 1 2 1 1 2 3 1 2 3 2 2 1 2 2 2 3 2 3 2 1 1 2 1 3 2 2 3 2 3 1 3 1 1 2 2 2 3 2 1 1 2 2 1 3 1 2 1 3 1 2 3 2 1 1 3 1 3 1 1 1 2 2 3 2 3 1 1 1 1 3 1 2 2 1 1 3 1 3 1 1 3 2 2 1 1 2 1 3 1 3 2 1 3 1 1 3 2 1 1 1 2 2 3 2 3 1 1 2 3 1 1 1 3 1 1 1 1 1 2 3 2 1 1 3 1 1 1 3 1 1 3 1 2 2 3 2 2 3 2 1 2 2 2 3 1 2 2 2 1 2 3 2 3 2 2 1 2 3 2 2 3 1 3 2 3 2 1 2 2 3 1 3 1 1 1 2 2 2 3 1 1 3 1 1 2 3 1 1 3 1 1 2 2 3 2 1 2 3 1 1 1 2 3 1 1 2 2 3 2 1 1 3 2 1 2 2 3 2 1 3 1 1 3 2 1 1 1 3 2 2 1 3 1 1 3 2 2 2 2 1 2 3 2 1 1 2 3 1 2 1 1 3 2 3 2 1 3 2 2 3 1 2 1 2 1 3 2 2 3 1 1 1 2 2 3 2 3 1 2 1 3 2 3 2 1 2 1 1 3 1 1 1 2 2 1 3 1 3 1 3 2 2 3 2 1 1 1 3 3 1 1 2 2 3 2 3 1 1 1 2 3 2 3 1 2 2 3 1 2 1 2 1 1 1 1 2 1 1 3 2 1 3 2 2 2 1 1 2 3 1 3 1 3 1 1 3 3 1 2 2 1 1 1 3 1 1 3 2 1 1 3 2 3 1 1 2 3 2 2 2 2 1 2 3 2 3 2 3 2 2 3 2 2 2 1 3 2 3 2 2 1 2 2 1 3 1 3 2 2 1 2 1 2 3 2 1 3 2 2 1 3 1 3 2 2 1 2 1 3 1 1 1 3 1 1 1 3 1 1 3 2 3 2 2 1 1 3 2 2 1 1 1 2 1 3 2 1 2 2 1 3 2 1 1 3 2 1 2 3 2 3 1 2 2 3 2 2 2 3 2 3 2 3 1 2 2 3 1 1 2 1 2 2 3 2 3 1 1 1 2 1 2 3 2 3 1 1 1 3 1 3 2 2 1 1 3 2 3 1 2 2 1 1 1 3 1 2 2 3 1 1 2 3 1 2 2 3 1 3 1 2 1 2 3 2 1 1 1 1 1 3 1 2 3 1 2 1 3 2 2 1 1 3 2 3 2 1 1 3 2 2 1 2 1 3 2 2 3 2 2 1 2 2 3 1 3 1 1 2 2 2 1 3 1 1 3 2 2 2 1 2 1 3 2 3 1 1 2 2 1 2 3 1 3 2 3 1 1 1 3 3 1 2 1 3 1 2 2 2 1 3 1 1 2 3 1 1 2 2 1 1 3 2 3 2 2 2 3 1 1 3 1 1 3 1 3 1 2 2 2 3 1 1 1 2 2 3 1 1 2 3 1 1 2 1 1 3 1 3 2 2 3 1 2 1 1 1 2 3 2 3 1 2 3 2 2 2 1 2 3 2 1 3 2 3 2 1 3 1 2 2 3 1 1 2 2 2 2 2 1 1 3 2 3 1 3 2 2 1 2 1 3 1 1 3 2 1 3 2 1 3 1 2 2 2 1 2 3 2 3 2 2 2 3 1 1 3 2 2 1 1 3 1 2 2 1 3 2 2 1 3 1 3 1 1 1 3 2 3 1 2 1 1 1 3 2 2 1 3 2 1 1 2 3 1 2 1 1 2 3 1 1 3 2 3 2 1 2 1 2 1 3 1 1 2 3 1 1 3 2 3 2 2 1 3 2 1 2 1 3 1 2 1 3 2 1 2 1 1 1 2 2 3 1 3 2 2 2 3 2 2 2 3 1 2 2 3 2 1 3 2 1 1 2 3 1 1 3 1 1 2 1 1 3 2 1 2 3 1 3 2 3 2 2 1 1 1 2 3 2 1 1 2 1 3 2 3 2 2 3 2 2 1 3 2 2 1 3 1 3 1 3 2 2 1 3 2 3 1 1 1 2 3 2 2 3 2 2 1 1 1 2 3 1 1 1 2 1 3 1 1 1 2 3 2 1 2 2 3 2 2 2 3 2 3 1 1 3 2 2 1 2 1 1 3 2 2 2 3 2 3 1 3 1 1 2 2 1 1 3 3 1 3 2 2 2 1 2 1 3 2 2 1 3 1 1 2 1 2 3 2 2 3 2 1 3 1 3 2 2 1 2 2 1 3 1 1 3 1 1 3 1 2 2 2 1 1 3 3 1 3 2 2 1 1 2 3 1 1 1 2 1 1 3 2 1 2 2 2 3 2 3 1 2 3 1 2 3 1 1 2 1 3 2 2 3 1 1 3 2 1 2 1 2 1 3 1 2 1 3 1 2 1 2 3 1 3 1 2 3 1 1 1 3 2 2 1 3 2 1 2 1 2 3 2 1 1 1 3 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2 2 3 2 2 3 2 1 1 1 3 1 3 1 2 3 2 1 1 1 3 2 3 1 3 2 2 1 2 2 1 2 2 3 1 1 3 1 1 1 3 2 2 1 3 1 2 3 1 2 3 1 1 2 2 1 2 3 2 2 1 2 2 2 3 2 2 2 1 3 2 2 2 3 2 3 2 3 1 1 1 1 2 1 2 3 1 1 2 2 2 3 1 1 3 1 3 1 1 3 2 3 1 3 1 2 2 1 3 1 2 1 2 1 3 1 1 2 1 2 2 3 1 1 3 1 3 2 2 1 3 1 1 2 1 1 3 1 3 1 1 1 2 3 2 1 2 3 2 3 2 2 2 2 1 2 3 1 1 1 3 1 3 1 1 3 2 3 2 1 2 2 1 2 3 3 2 1 1 3 2 1 2 2 1 1 3 2 3 2 3 1 2 2 2 1 3 2 1 1 2 1 1 1 3 1 3 1 1 3 2 1 1 1 3 1 3 2 1 1 1 3 2 1 2 2 3 2 2 1 1 2 2 3 1 1 3 2 3 2 1 2 3 1 1 1 3 2 1 2 1 2 1 3 2 2 3 1 3 2 2 3 1 3 2 1 1 3 1 2 2 2 1 3 1 2 3 1 3 1 2 1 2 1 2 3 1 1 1 3 1 2 1 3 2 1 2 1 1 3 1 1 3 1 2 3 1 2 2 2 3 2 3 2 1 1 1 2 3 2 2 1 3 2 1 1 2 1 1 3 1 1 1 3 1 2 3 1 1 3 1 3 1 3 1 2 2 2 3 2 3 1 3 2 1 1 1 3 2 1 1 1 2 1 3 1 1 1 1 1 2 1 3 1 2 3 2 1 3 1 1 2 2 2 3 2 3 2 3 2 2 3 1 1 1 2 2 1 3 2 3 2 2 2 3 2 3 2 3 2 1 2 2 1 2 1 2 2 2 3 1 3 2 1 2 3 1 2 1 3 1 1 3 1 2 2 3 2 2 1 2 1 3 1 3 2 2 3 1 1 3 2 1 2 3 2 1 1 1 3 2 2 2 2 1 1 2 2 2 3 2 3 1 1 2 3 2 2 3 2 2 1 2 2 3 2 3 2 2 1 3 2 2 2 1 2 3 1 3 1 3 2 3 1 3 1 2 2 2 1 1 3 2 2 3 2 2 1 3 1 3 2 3 2 2 2 1 2 3 1 1 1 2 2 2 2 2 2 1 2 2 3 2 3 1 2 3 2 3 1 1 1 2 1 1 3 1 3 1 3 2 1 1 3 2 1 1 2 1 2 3 1 2 1 3 2 3 1 2 2 1 1 3 2 3 1 3 1 2 3 1 2 3 2 1 2 1 2 3 2 1 3 2 1 1 2 1 1 1 2 2 3 1 3 1 1 1 3 1 3 1 2 1 1 1 2 3 1 2 1 3 2 2 2 3 1 1 3 2 3 1 2 3 2 2 1 3 1 1 2 3 2 2 2 1 1 3 2 2 3 2 2 3 2 3 2 1 1 2 2 3 2 2 1 3 2 1 1 1 1 2 1 3 2 3 1 3 1 1 3 2 3 1 2 1 1 3 1 2 1 2 2 2 3 2 3 2 3 1 2 1 1 3 2 1 1 2 2 3 1 3 2 2 1 1 1 2 2 1 3 2 2 1 2 2 3 2 2 2 3 2 3 1 1 2 2 2 3 1 3 1 1 2 1 3 2 2 3 1 1 2 1 3 2 1 1 2 2 2 3 1 1 3 1 3 1 2 3 2 2 2 3 2 3 2 2 2 3 1 1 2 1 3 1 3 1 1 2 1 2 3 1 2 1 1 1 3 1 2 1 2 3 1 3 1 3 1 2 2 3 2 1 1 2 1 1 1 3 1 2 3 1 3 1 2 3 2 2 3 2 2 1 1 1 3 2 2 1 1 3 2 3 1 1 1 2 2 2 3 2 1 1 3 1 1 2 2 1 3 2 3 3 1 1 1 2 3 1 3 1 3 2 2 1 2 2 3 1 2 1 3 2 2 2 1 2 2 2 3 2 1 1 1 2 3 1 3 1 2 1 2 1 3 2 3 2 2 1 3 3 2 2 1 1 2 2 3 2 3 1 2 1 2 2 2 3 1 2 2 1 3 2 3 1 3 1 3 2 3 2 2 3 1 2 1 1 1 3 1 2 3 2 2 2 1 2 1 1 1 2 2 3 2 3 1 3 1 1 1 2 2 3 1 2 1 1 3 1 1 3 1 2 2 1 1 1 3 1 3 1 1 2 2 3 1 3 1 1 3 1 3 1 1 1 2 2 2 3 2 2 1 3 1 1 3 1 1 2 2 3 1 1 2 3 2 1 2 3 2 1 3 2 2 1 1 3 1 2 1 2 3 2 3 2 3 1 2 3 2 2 2 1 1 2 3 1 3 2 2 1 2 3 2 2 3 2 1 1 2 1 3 1 1 1 2 2 3 2 2 1 3 1 2 1 1 3 2 2 2 1 3 1 3 1 2 2 3 1 3 1 1 1 2 3 1 3 2 1 1 2 1 1 3 1 3 2 1 2 2 2 3 1 1 3 2 2 3 2 2 2 1 1 3 2 3 2 1 1 2 3 1 2 2 2 3 2 2 1 3 2 2 3 1 1 3 1 1 3 1 2 2 3 2 2 1 2 2 3 2 2 3 1 1 2 1 2 1 3 1 1 1 3 1 2 2 1 1 1 3 1 3 2 3 1 1 2 3 2 1 1 1 2 2 3 2 2 1 3 1 1 1 2 2 2 3 1 3 2 3 2 3 1 2 2 3 2 2 1 3 2 3 2 3 2 2 1 2 2 3 1 2 2 1 2 3 3 1 3 1 1 2 2 1 2 3 2 3 2 3 1 1 2 1 2 1 3 1 1 1 2 2 3 1 2 2 3 1 2 1 1 1 3 2 1 1 1 3 1 3 2 3 2 1 3 2 3 2 3 2 1 1 1 2 2 3 1 1 2 1 2 3 2 2 1 1 2 3 1 1 1 3 2 1 1 1 3 1 1 1 3 1 1 3 2 2 2 3 1 1 1 3 2 2 2 1 3 2 2 3 1 1 3 1 1 2 1 3 1 1 1 3 1 1 1 3 3 2 3 2 1 1 2 1 1 3 1 3 2 3 1 1 2 1 3 2 1 1 2 2 2 1 2 2 3 1 1 1 2 1 1 3 1 3 1 3 1 2 2 2 3 2 3 1 1 2 3 1 3 1 1 1 3 1 1 3 1 1 3 2 2 1 1 3 1 2 2 2 1 1 3 1 3 2 3 1 3 2 1 2 1 2 2 3 2 2 1 1 1 3 1 1 2 2 2 3 2 1 1 1 3 2 3 1 2 3 1 2 3 2 1 1 3 1 2 1 3 1 2 3 2 2 1 2 3 2 3 1 2 3 1 1 1 2 1 2 3 2 1 2 3 2 1 3 1 1 2 1 1 1 3 2 3 2 2 1 1 1 3 2 3 2 2 1 1 1 1 3 1 3 2 1 2 3 2 3 2 3 2 1 2 3 1 2 1 2 2 2 1 1 1 3 1 2 1 1 3 1 3 2 2 1 3 2 1 1 1 2 2 3 2 3 1 1 3 1 1 2 2 1 3 1 3 1 1 2 1 1 3 2 3 2 3 1 2 1 3 1 2 1 1 3 1 1 1 3 2 3 1 1 1 2 3 2 1 1 1 2 2 3 3 1 2 3 1 1 1 3 1 2 3 2 2 2 1 1 1 3 2 2 2 3 2 2 1 3 2 3 2 1 1 3 2 1 1 2 1 1 3 2 2 2 3 1 3 1 1 1 3 2 2 3 1 3 1 1 2 2 1 3 1 1 2 2 2 3 1 2 1 1 1 3 2 2 1 1 3 1 1 1 2 2 2 3 2 1 2 3 2 3 2 2 3 2 2 3 1 3 1 1 3 1 2 2 2 1 3 1 2 3 1 1 1 2 3 1 3 2 2 2 2 1 1 3 2 2 2 3 1 3 1 2 1 1 1 3 1 2 3 1 2 1 2 3 2 3 1 3 1 2 1 3 2 2 2 3 2 1 1 2 1 2 3 2 2 2 3 2 1 3 2 2 2 3 1 1 1 2 2 3 2 1 1 3 2 2 2 3 1 2 3 1 2 1 3 1 1 2 2 3 1 2 2 1 1 2 3 1 2 3 1 3 2 1 3 2 1 3 1 3 1 2 2 2 3 2 1 1 2 1 1 3 2 1 2 2 3 1 1 3 1 2 1 1 2 3 1 2 3 2 1 1 2 3 2 1 1 3 2 1 3 2 3 2 2 3 1 1 1 2 2 2 3 1 2 3 1 3 1 3 1 2 1 2 3 2 2 1 2 3 2 3 2 1 1 1 3 2 1 2 1 3 2 2 2 1 2 3 2 2 1 3 2 3 1 1 2 1 1 3 2 3 1 1 1 2 1 3 1 1 2 3 1 1 2 3 1 1 1 3 1 1 1 3 1 2 2 3 2 1 1 2 1 1 3 2 1 3 1 3 1 1 2 3 1 1 1 2 1 3 2 3 2 2 1 1 1 2 3 1 3 2 3 2 3 2 1 3 1 2 1 1 1 3 1 2 3 2 3 1 1 2 2 1 2 3 1 2 3 1 2 1 3 2 1 2 1 2 3 2 3 2 3 2 1 2 2 2 3 2 2 2 1 2 3 2 2 2 3 2 1 3 1 1 1 2 3 2 2 2 3 1 1 3 1 2 1 1 1 2 2 2 3 2 1 3 1 3 1 3 1 1 1 2 2 2 3 2 2 3 2 1 3 1 1 2 1 1 3 1 2 2 1 3 2 1 1 3 2 3 2 1 3 1 2 3 1 2 2 2 1 3 1 3 1 1 1 2 1 2 3 1 3 2 1 3 1 1 1 1 2 1 3 1 3 2 1 2 3 2 2 3 2 2 2 1 2 3 1 3 1 1 3 1 2 3 1 2 3 1 1 3 1 3 2 2 2 1 2 2 3 2 1 1 1 2 1 1 3 2 1 1 1 3 1 1 3 1 1 3 1 1 1 2 3 2 3 2 2 1 2 2 3 1 1 3 1 1 2 2 1 1 3 2 3 2 2 2 1 3 2 3 2 1 1 3 1 1 1 3 1 1 2 3 2 2 3 1 2 2 2 1 2 3 1 2 3 2 3 1 2 2 1 1 1 3 1 3 1 2 3 2 2 3 1 2 2 3 1 1 1 2 1 1 2 3 1 2 1 1 2 2 3 2 2 3 1 3 1 3 1 3 2 1 1 2 3 2 2 2 3 2 2 3 1 1 1 3 2 3 2 1 1 1 3 2 1 2 1 2 2 3 1 3 2 2 1 2 1 2 3 1 3 1 1 1 3 2 3 2 1 1 2 2 2 2 3 2 3 1 3 1 1 1 3 1 1 3 2 1 2 1 2 1 3 1 1 2 3 2 1 1 3 2 2 2 1 3 1 3 2 2 1 2 1 3 1 3 2 2 2 1 3 1 2 1 3 1 2 1 3 1 2 1 1 3 2 2 1 1 2 2 3 1 1 3 1 3 1 3 1 2 3 1 2 2 3 2 2 2 1 2 3 2 1 2 2 1 2 3 1 1 1 3 2 2 1 1 3 1 1 1 2 2 3 2 1 3 2 3 1 2 1 3 2 2 2 3 1 2 1 2 2 3 2 2 2 3 2 3 1 3 2 3 2 1 2 1 1 2 2 2 3 2 1 3 1 1 1 3 2 2 3 2 2 1 2 3 1 3 2 2 3 1 2 2 2 3 1 3 2 1 1 3 2 2 2 1 2 1 3 1 2 3 1 1 1 1 3 1 2 1 1 1 3 2 3 1 3 2 2 3 1 2 2 2 1 3 1 2 3 1 2 1 2 2 3 2 1 1 3 1 2 1 2 3 2 2 3 2 1 1 1 3 3 2 1 1 3 1 3 2 3 2 1 2 2 3 2 1 1 3 2 2 1 1 2 2 3 2 3 2 3 1 2 2 1 3 2 1 1 2 3 1 1 3 2 1 2 2 2 1 3 2 1 1 3 1 1 1 3 1 2 2 1 1 3 2 3 2 2 1 3 2 1 1 1 3 2 1 3 1 1 1 3 2 2 3 1 1 1 2 2 3 1 2 2 1 2 3 2 1 1 3 1 3 1 1 3 2 2 3 1 3 2 1 1 2 3 2 1 2 2 2 3 2 2 1 1 3 1 1 1 2 1 3 2 1 3 1 2 1 1 3 2 3 1 1 2 1 1 3 2 1 1 1 2 2 3 1 1 1 3 2 3 2 1 2 1 3 2 3 1 1 3 1 2 3 2 1 2 3 2 2 2 1 2 2 3 2 2 3 2 3 2 1 1 2 2 2 1 3 1 1 2 1 2 1 3 2 3 1 1 3 1 3 1 2 1 3 3 2 2 1 2 3 1 1 1 3 1 3 2 1 2 3 2 3 2 2 1 1 1 2 2 1 2 2 1 2 3 2 3 1 1 3 1 1 3 1 1 2 3 1 2 2 1 3 2 1 1 2 1 1 3 2 2 3 1 1 3 1 3 1 1 2 2 3 2 2 3 2 2 3 1 2 3 2 2 2 3 1 2 3 2 1 1 2 2 3 2 2 1 1 1 3 3 2 3 1 1 1 3 1 2 2 2 3 1 3 2 2 2 3 2 1 2 1 1 2 1 3 1 3 1 1 2 1 2 1 3 1 2 2 3 1 3 1 2 2 2 3 2 2 2 2 2 3 1 3 1 2 3 2 3 1 2 3 1 2 1 1 1 3 2 2 1 1 3 2 2 3 2 1 1 1 2 2 3 2 1 3 2 1 1 1 3 1 1 3 2 1 3 2 3 2 2 1 2 3 1 2 3 2 2 3 2 2 2 3 2 1 2 2 1 2 1 2 2 1 2 2 3 2 3 2 1 3 1 2 3 2 1 2 2 1 1 3 1 3 3 2 2 1 3 1 1 1 3 1 2 2 2 1 3 1 1 3 2 2 1 3 2 2 2 2 3 2 3 2 1 2 2 1 1 3 1 3 1 3 2 3 1 1 1 2 1 2 3 2 2 2 1 1 3 1 2 1 3 1 1 1 3 1 3 2 3 1 2 2 2 1 1 1 2 3 1 3 1 1 1 2 1 3 1 2 1 3 2 2 1 2 2 3 2 3 2 3 1 1 2 2 3 1 1 2 1 1 3 1 1 2 2 2 3 2 2 3 2 3 1 1 2 1 1 3 1 2 2 3 1 1 2 2 1 3 2 3 1 3 2 1 1 3 1 1 2 3 2 2 2 3 1 3 1 3 1 2 2 2 1 3 2 1 1 1 3 1 1 3 2 2 2 1 3 1 1 2 1 3 1 1 1 2 3 2 3 2 2 2 3 1 2 1 2 1 1 3 2 1 1 3 2 3 2 2 1 1 3 1 2 2 2 3 1 3 3 2 1 3 2 3 1 1 2 1 1 3 2 2 1 3 2 3 2 2 1 1 2 1 1 1 3 2 3 2 3 2 2 1 1 1 3 2 1 1 1 2 3 2 1 3 1 2 1 3 1 3 1 2 3 2 2 2 1 2 3 2 2 3 2 3 1 1 2 2 1 1 1 3 2 2 3 1 1 2 1 2 2 3 1 2 3 1 2 1 1 3 1 1 3 1 2 3 1 1 2 3 2 3 1 3 1 2 3 2 2 2 1 3 1 1 2 1 1 2 1 1 2 1 1 2 3 1 2 3 2 1 1 3 2 2 2 3 1 3 2 2 2 3 1 1 1 3 1 3 2 3 1 1 2 1 3 1 1 1 2 1 1 3 1 3 1 1 1 1 2 1 1 1 3 2 2 1 2 2 3 1 3 1 3 1 3 2 2 2 1 3 1 3 2 1 3 2 3 2 2 3 2 1 3 2 2 2 1 3 2 1 2 1 2 1 3 2 1 1 3 1 1 2 3 2 1 2 2 1 3 1 2 1 2 2 2 3 2 3 3 1 2 1 1 1 2 3 2 2 2 3 1 2 1 1 1 3 2 1 3 2 2 3 1 2 1 3 2 1 2 3 2 1 2 3 2 3 2 3 1 1 3 1 2 2 2 1 1 2 3 1 1 2 3 2 1 3 1 3 2 3 1 2 2 1 3 2 2 2 1 1 3 2 1 3 2 1 2 2 2 1 3 2 3 1 2 3 2 1 1 3 1 1 2 1 1 3 1 1 2 2 3 2 1 2 2 3 1 1 3 1 1 3 1 1 2 1 2 3 2 2 2 1 2 1 3 1 1 2 2 3 1 3 1 3 1 1 3 2 2 1 1 3 1 1 1 3 2 1 3 2 1 3 1 3 1 2 2 2 3 1 3 1 1 2 2 1 2 2 2 1 1 1 3 1 1 1 3 2 1 2 2 3 2 1 1 3 1 3 2 3 1 1 1 2 2 3 1 3 1 1 1 3 2 3 1 1 2 3 1 1 3 2 2 2 1 1 3 1 1 1 2 1 1 3 2 1 2 3 1 2 1 3 2 1 3 2 1 3 1 2 2 2 3 1 1 2 2 3 2 1 2 2 3 2 1 3 2 2 2 3 2 3 1 1 3 1 3 1 1 2 1 1 2 3 2 1 3 1 3 1 2 1 2 1 1 3 2 3 2 3 2 1 1 2 1 3 2 2 3 2 2 1 1 2 3 1 3 2 1 1 2 1 2 1 3 2 2 3 2 1 3 2 2 2 1 3 1 2 3 1 1 2 3 2 1 2 2 3 2 3 2 2 1 3 1 1 2 3 1 2 3 2 2 1 1 2 1 3 3 2 2 2 3 2 1 2 1 3 2 1 2 2 2 3 1 2 2 3 1 2 3 2 1 3 1 3 2 1 1 1 3 2 1 2 3 1 3 2 2 1 2 3 1 1 2 1 3 1 1 1 3 2 2 2 1 1 3 2 3 1 2 3 2 1 2 1 2 2 3 2 2 2 1 1 1 2 3 1 2 1 1 1 3 1 3 2 1 3 2 3 1 1 3 2 2 2 1 1 1 2 3 2 3 2 3 1 3 1 1 3 1 2 3 1 1 2 1 1 1 2 2 2 3 2 1 2 1 1 1 3 2 3 1 1 3 1 1 3 1 3 1 1 2 3 1 2 2 1 3 2 1 2 2 2 3 2 3 1 1 3 1 3 1 2 2 2 2 2 2 3 1 1 2 3 1 1 1 2 2 3 1 2 3 1 2 1 3 1 2 3 1 3 1 3 2 1 1 3 1 2 2 1 1 3 1 1 2 1 1 3 1 1 1 3 1 2 2 3 1 1 2 2 3 1 3 1 1 3 2 3 1 1 3 2 1 1 1 2 2 2 2 1 3 1 3 1 1 3 2 1 2 2 3 2 2 2 3 1 1 1 3 1 2 1 1 1 3 2 3 1 1 1 3 1 2 2 2 3 1 1 1 2 3 1 2 3 3 1 1 1 3 2 2 1 3 1 3 1 1 1 2 3 2 1 3 1 1 1 2 2 3 2 3 1 1 2 1 1 2 3 1 1 3 1 1 3 2 2 1 2 3 2 2 1 2 2 3 2 3 1 1 2 1 1 1 3 2 1 3 1 2 3 2 3 2 2 1 2 2 2 1 2 1 2 3 1 2 1 2 3 1 3 2 2 2 3 2 3 2 2 3 1 2 2 3 1 2 2 2 3 2 3 2 3 1 3 2 1 2 2 1 3 2 2 1 2 1 1 1 3 2 3 1 2 2 1 1 3 2 2 1 3 2 2 2 3 1 3 1 2 2 2 3 2 1 2 2 2 3 2 1 2 1 1 2 3 2 2 3 1 1 3 1 3 3 2 2 2 3 1 1 1 2 2 1 3 2 3 2 3 1 3 1 1 1 2 1 2 1 1 2 3 2 2 3 1 3 1 2 2 3 1 2 1 1 2 3 2 2 3 1 1 2 1 3 2 1 3 2 1 3 2 1 2 2 3 2 2 3 2 1 1 2 1 1 3 3 2 2 3 2 1 1 2 2 2 3 1 3 2 3 2 2 1 3 2 2 1 2 2 2 3 1 1 2 1 2 3 1 2 1 3 2 2 1 3 2 1 1 2 2 3 2 3 2 3 1 2 1 3 2 1 2 3 2 2 2 3 2 3 1 2 2 1 1 1 3 1 3 1 2 3 2 1 2 1 1 1 3 1 3 2 1 2 3 2 2 1 2 1 1 3 1 3 2 3 1 3 1 2 2 2 1 3 1 1 3 1 2 3 2 2 1 2 2 1 1 2 3 1 3 1 1 2 2 2 3 2 2 1 1 1 3 1 3 1 1 1 3 2 1 1 1 3 1 1 2 2 1 3 2 1 2 3 1 2 1 3 1 2 3 1 3 1 2 1 3 1 3 2 2 3 2 1 2 1 3 2 2 2 1 2 1 3 2 2 3 1 3 2 1 3 1 1 2 3 1 2 2 3 2 2 2 1 3 1 1 3 1 2 2 2 3 2 2 2 1 2 3 2 2 2 3 1 3 1 1 3 1 3 2 2 1 2 2 2 2 1 3 1 1 3 2 2 2 3 1 1 1 3 2 2 1 2 2 3 1 2 2 3 3 1 2 3 1 1 3 1 3 2 1 2 2 2 3 2 2 1 2 1 2 3 2 1 3 1 2 3 1 1 2 1 2 1 3 2 1 1 3 2 1 2 2 3 1 3 2 1 2 1 3 2 3 1 2 3 1 1 1 2 2 2 3 1 3 1 2 1 3 1 2 1 3 1 1 1 3 1 1 1 2 2 3 1 1 3 1 3 2 2 2 3 1 2 1 2 1 2 2 2 3 1 3 2 1 2 2 2 3 2 3 2 1 2 2 3 1 1 2 3 1 2 3 1 3 2 2 3 1 1 1 2 2 2 3 1 1 3 2 1 2 2 3 2 2 2 1 1 2 1 3 2 3 1 3 1 3 1 3 2 1 2 1 2 3 2 1 1 1 2 2 1 1 3 1 3 1 3 2 3 1 3 2 1 1 1 2 3 2 1 1 1 1 1 3 1 1 2 1 3 1 2 3 1 3 1 2 2 1 3 1 1 1 2 1 3 1 3 2 2 2 1 1 1 3 1 3 2 2 1 3 1 1 2 2 3 1 1 1 3 3 2 1 1 3 1 2 2 2 3 2 2 3 1 1 2 1 1 1 3 1 1 3 1 1 3 1 3 1 1 1 3 1 1 3 2 2 1 1 1 3 2 3 1 2 1 2 2 2 1 1 2 1 3 1 3 1 1 3 1 3 1 2 3 2 1 2 3 1 1 2 1 2 2 1 2 2 1 3 2 3 1 2 1 1 3 2 3 1 1 3 2 2 2 1 3 1 2 1 1 2 3 2 1 1 1 3 1 2 3 1 3 2 2 2 1 2 3 1 3 2 2 3 1 2 2 2 3 1 3 1 3 2 2 3 1 2 1 1 3 1 2 2 2 1 2 3 1 2 2 1 2 2 3 2 3 2 3 2 1 3 1 1 2 2 1 3 1 2 1 2 1 1 1 3 1 2 1 2 1 3 2 1 3 1 2 3 1 2 3 2 3 2 2 2 1 3 2 2 3 1 3 1 2 3 1 1 3 2 2 1 2 2 1 3 1 1 2 2 3 1 1 2 2 3 1 2 1 2 1 3 2 3 2 1 1 1 3 2 3 3 1 1 3 1 1 1 3 1 2 2 1 2 2 3 2 1 2 2 3 1 3 2 2 1 2 2 3 1 3 2 3 2 1 3 2 3 1 2 2 2 1 3 1 1 1 2 1 1 1 2 1 1 1 3 2 3 2 2 2 1 1 3 1 3 2 1 3 1 3 2 1 3 2 1 3 1 3 1 2 1 1 2 2 3 1 2 3 2 3 2 1 1 2 2 2

wherein each of 1 to 3 is a nucleotide base selected to be different from the others of 1 to 3 with the proviso that up to three nucleotide bases of each sequence can be substituted with any nucleotide base provided that: for any pair of sequences of the set: M1≦16, M2≦13, M3≦20, M4≦16, and M5≦19 where: M1 is the maximum number of matches for any alignment in which there are no internal indels; M2 is the maximum length of a block of matches for any alignment; M3 is the maximum number of matches for any alignment having a maximum score; M4 is the maximum sum of the lengths of the longest two blocks of matches for any alignment of maximum score; and M5 is the maximum sum of the lengths of all the blocks of matches having a length of at least 3, for any alignment of maximum score; wherein: the score of an alignment is determined according to the equation (A×m)−(B×mm)−(C×(og+eg))−(D×eg)), wherein: for each of (i) to (iv):  (i) m=6, mm=6, og=0 and eg=6,  (ii) m=6, mm=6, og=0 and eg=1,  (iii) m=6, mm=2, og=5 and eg=1, and  (iv) m=6, mm=6, og=6 and eg=0, A is the total number of matched pairs of bases in the alignment; B is the total number of internal mismatched pairs in the alignment; C is the total number of internal gaps in the alignment; and D is the total number of internal indels in the alignment minus the total number of internal gaps in the alignment; and wherein the maximum score is determined separately for each of (i), (ii), (iii) and (iv). v) a second target nucleic acid, distinct from said first target nucleic acid, and having a fourth region, a fifth region and a sixth region, wherein said fourth region is located adjacent to and downstream from said fifth region, and said fifth region is located adjacent to and downstream from said sixth region, said fifth region having a sequence complementary to said 3′ portion of said sequence selected from the group of sequences listed in step (a)(iv), said sixth region having a sequence complementary to said 5′ portion of the sequence selected from the group of sequences in step (a)(iv); vi) a third oligonucleotide having a 5′ portion, a central portion and a 3′ portion, said 5′ portion of said third oligonucleotide having a sequence complementary to said fourth region of said second target nucleic acid, said central portion of said third oligonucleotide having a sequence complementary to said fifth region of said second target nucleic acid, and said 3′ portion of said third oligonucleotide having a sequence that is not base paired to either said second target nucleic acid or said first target nucleic acid and is selected from a set of oligonucleotides based on the group of sequences listed in step (a)(iv) such that said sequence selected is distinct from said sequence selected in step (a)(iv); b) mixing said cleavage means, said first target nucleic acid, said second target nucleic acid, said first, second, and third oligonucleotides to create a reaction mixture under reaction conditions such that at least said 5′ portion of said first oligonucleotide is annealed to said first target nucleic acid and wherein at least said 5′ and central portion of said second oligonucleotide is annealed to said first target nucleic acid so as to create a first cleavage structure and wherein the combined melting temperature of said complementary regions within said 5′ and 3′ portions of said first oligonucleotide when annealed to said first target nucleic acid is greater than the melting temperature of said 5′ and central portion of said first oligonucleotide, wherein cleavage of said first cleavage structure occurs to generate a first non-target cleavage product, and wherein at least said 5′ portion first non-target cleavage product is annealed to said second target nucleic acid and at least said 5′ and central portion of said third oligonucleotide is annealed to said second target nucleic acid so as to create a second cleavage structure and wherein the combined melting temperature of said complementary regions within said 5′ and 3′ portions of said non-target cleavage product when annealed to said second target nucleic acid is greater than the melting temperature of said 5′ and central portion of said third oligonucleotide, wherein cleavage of said second cleavage structure occurs to generate a second non-target cleavage product; and c) detecting said second non-target cleavage product.
 68. The method of claim 67, wherein said first target nucleic acid is genomic DNA and said second target nucleic acid is synthetic DNA.
 69. The method of claim 68, wherein said synthetic DNA has at least one hairpin loop.
 70. The method of claim 69, wherein the method includes a plurality of said first target nucleic acid sequences, a plurality of first oligonucleotide molecules, a plurality of said second oligonucleotide molecules, a plurality of said second target nucleic acid sequences and a plurality of third oligonucleotide molecules. 